Immunomodulatory properties of multipotent adult progenitor cells and uses thereof

ABSTRACT

Isolated cells are described that are not embryonic stem cells, not embryonic germ cells, and not germ cells. The cells can differentiate into at least one cell type of each of at least two of the endodermal, ectodermal, and mesodermal lineages. The cells do not provoke a harmful immune response. The cells can modulate immune responses. As an example, the cells can suppress an immune response in a host engendered by allogeneic cells, tissues, and organs. Methods are described for using the cells, by themselves or adjunctively, to treat subjects. For instance, the cells can be used adjunctively for immunosuppression in transplant therapy. Methods for obtaining the cells and compositions for using them also are described.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/269,736 filed 9 Nov. 2005.

STATEMENT OF GOVERNMENT RIGHTS

Not Applicable.

FIELD OF THE INVENTION

The field of the invention is immunomodulation by multipotent adultprogenitor cells (“MAPCs”) and their use for modulating immune responsesin primary and adjunctive therapies.

BACKGROUND OF THE INVENTION

The therapeutic use of organ transplants, including bone marrowtransplants, has steadily increased since its early beginnings. It hasbecome an important therapeutic option for a number of diseases,including, but not limited to, hematologic, immunologic, and malignantdisorders.

Unfortunately, the therapeutic uses of transplantation often arecomplicated, rendered ineffectual, or precluded by adverse immuneresponses engendered by the transplant. Among the most prominent adversereactions encountered as a result of transplant therapies are (i) thehost versus graft response (“HVG”) (rejection of the transplant by animmune competent host), and (ii) graft versus host disease (“GVHD”)(processes that occur primarily in an immunocompromised host when it isrecognized as non-self by immunocompetent cells of a graft).

Graft rejection in a host can be avoided, of course, by perfectlymatching the donor and the host. Except for autologous tissue, however,only identical twins might be expected to be truly syngeneic. Perfectmatches between an individual donor and another individualhost/recipient are virtually non-existent. Thus, the use of autologoustissue is the only other way to make a perfect match. Unfortunately, thehost tissue is typically not suitable or was not isolated in advance ofneed. Frequently the need for the transplant therapy is, in fact, toreplace damaged tissue in the host. The use of syngeneic tissue,therefore, while an effective solution to the problems of adverse hostresponse to graft tissue, is not generally useful in practicalapplications.

If syngeneic matching is not possible, the adverse immune effects thatarise in transplant therapies can be mitigated by matching an allogeneicdonor and host as closely as possible. Blood and/or tissue typing isused to match donors and hosts to provide the highest likelihood oftherapeutic success. Even the closest matching of allogeneic tissue,however, does not prevent serious HVG, and, accordingly, transplanttherapies involve the use of immunosuppression and immunosuppressivedrugs, as discussed below.

Another approach to avoid the complications of HVG in transplanttherapies has been to disable the immune system of the recipient host.This has been accomplished by using radiation therapy, and/orimmunosuppressive chemotherapy, and/or antibody therapy. The resultingsuppression of host immune responses often quite effectively aidsestablishment of the graft (such as bone marrow) in the host. However,immunoablation or suppression compromises the host's immune defenses.This results in the host becoming all too readily susceptible toinfections after even minor exposure to infectious agents. The resultinginfections are a major cause of morbidity and mortality among transplantpatients.

Compromising the host immune system also engenders or exacerbatesanother serious problem encountered in transplant therapies—graft versushost disease (“GVHD”). GVHD occurs when donor tissue containsimmunocompetent cells that recognize MHC proteins of the recipient asnon-self. This activates the T-cells, and they secrete cytokines, suchas IL-2 (interleukin 2), IFN-γ (interferon γ), and TNF-α (tumor necrosisfactor α). These signals trigger an immune attack on recipient targetsincluding the skin, GI tract, liver, and lymphoid organs (Ferrara andDeeg, 1991). GVHD is particularly a problem in bone marrow transplants,where it has been shown to be mediated primarily by T lymphocytes (Grebeand Streilein, 1976). In fact, approximately 50% of bone marrowtransplant patients develop acute GVHD. Many of these patients die (from15% to 45%).

There are also other immune system dysfunctions, disorders, and diseasesthat arise as primary pathologies and as secondary effects of otherpathologies and/or treatments thereof. These include neoplasms,pathologies of the bone marrow, pathologies of the blood, autoimmunedisorders, and some inflammatory disorders, as discussed further below.Primary and adjunctive therapy for these disorders and diseases, likeprimary and adjunctive therapies for HVG and GVHD, often involve the useof immunosuppressive drugs. All of the current therapies havedisadvantages and side effects.

Immunosuppressant Drugs

A good deal of effort has been directed to developing drugs to treatthese immune system dysfunctions to ameliorate or eliminate theirdeleterious effects, without causing additional harmful side effects.There has been some progress toward this goal, and a number of drugshave been developed and are in use to prevent and/or treat thesedysfunctions. The introduction of the more effective of these drugsmarked a great advance in the medical practice of transplant therapies;but, none are ideal. Indeed, none of the immunosuppressive drugscurrently available for clinical use in transplant therapies areentirely effective. All of the drugs have serious drawbacks anddeleterious side effects, as summarized briefly below. For review seeFarag (2004), “Chronic graft-versus-host disease: where do we go fromhere?,” Bone Marrow Transplantation 33: 569-577.

Corticosteroids, which are used primarily to treat inflammation andinflammatory diseases, are known to be immunosuppressive and areconsidered by many to be the best primary treatment for HVG and GVHD.They inhibit T-cell proliferation and T-cell dependent immune responses,at least in part, by inhibiting the expression of certain cytokine genesinvolved in T-cell activation and T-cell dependent immune response.

Cyclosporin is among the most frequently used drugs for immunesuppression and the prevention of HVG and GVHD. It is stronglyimmunosuppressive in general. Although it can be effective in reducingadverse immune reactions in transplant patients, it also weakens theimmune system so much that patients are left highly vulnerable toinfections. Consequently, patients are much more easily infected byexposure pathogens, and have little capacity to mount an effectiveimmune response to infections. Even mild pathogens then can belife-threatening. Cyclosporin also causes a variety of other undesirableside effects.

Methotrexate is also widely used in the prophylaxis and treatment of HVGand GVHD, by itself or in combination with other drugs. Studies haveshown that, if it is effective at all, it is apparently less effectivethan cyclosporin. As with cyclosporin, methotrexate causes a variety ofside effects, some of which can be deleterious to patient health.

FK-506 is a macrolide-like compound. Similar to cyclosporin, it isderived from fungal sources. The immunosuppressive effects ofcyclosporin and FK-506 are similar. They block early events of T-cellactivation by forming a heterodimeric complex with their respectivecytoplasmic receptor proteins (i.e., cyclophilin and FK-bindingprotein). This then inhibits the phosphatase activity of calcineurin,thereby ultimately inhibiting the expression of nuclear regulatoryproteins and T-cell activation genes.

Other drugs that have been used for immunosuppression includeantithymocyte globulin, azathioprine, and cyclophosphamide. They havenot proven to be advantageous. Rapamycin, another macrolide-likecompound which interferes with the response of T-cells to IL-2, also hasbeen used to block T-cell activated immune response. RS-61443, aderivative of mycophenolic acid, has been found to inhibit allograftrejection in experimental animals. Mizoribine, an imidazole nucleoside,blocks the purine biosynthetic pathway and inhibits mitogen stimulatedT- and B-cell proliferation in a manner similar to azathioprine andRS-61443. Deoxyspergualin, a synthetic analog of spergualin, has beenfound to exert immunosuppressive properties in pre-clinicaltransplantation models. The anti-metabolite brequinar sodium is aninhibitor of dihydro-orotate dehydrogenase and blocks formation of thenucleotides uridine and cytidine via inhibition of pyrimidine synthesis.Berberine and its pharmacologically tolerable salts have been used as animmunosuppressant for treating autoimmune diseases such as rheumatism,for treating allergies, and for preventing graft rejection. It has beenreported that berberine inhibits B-cell antibody production andgenerally suppresses humoral immune responses, but does not affectT-cell propagation. See Japanese Patent 07-316051 and U.S. Pat. No.6,245,781.

None of these immunosuppressive drugs, whether used alone or incombination with other agents, are fully effective. All of themgenerally leave patients still susceptible to HVG and GVHD and weakentheir ability to defend against infection. This renders them much moresusceptible to infection and much less able to fight off infections whenthey do occur. Furthermore, all of these drugs cause serious sideeffects, including, for instance, gastrointestinal toxicity,nephrotoxicity, hypertension, myelosuppression, hepatotoxicity,hypertension, and gum hypertrophy, among others. None of them haveproven to be a fully acceptable or effective treatment. In sum, giventhese drawbacks, there is at present no entirely satisfactorypharmaceutically based treatment for adverse immune system dysfunctionand/or responses such as HVG and GVHD.

It has long been thought that a more specific type of immune suppressionmight be developed without these drawbacks. For example, an agent thatsuppressed or eliminated alloreactive T-cells, specifically, would beeffective against HVG and GVHD (at least for allogeneic grafts) withoutthe deleterious side effects that occur with agents that globally attackand compromise the immune system. However, as yet, no such agent(s) havebeen developed.

Use of Restricted Stem Cells in Transplantation

The use of stem cells in lieu of or together with immunosuppressiveagents has recently attracted interest. There have been some encouragingobservations in this area. A variety of stem cells have been isolatedand characterized in recent years. They range from those of highlyrestricted differentiation potential and limited ability to grow inculture to those with apparently unrestricted differentiation potentialand unlimited ability to grow in culture. The former have generally beenthe easier to derive and can be obtained from a variety of adulttissues. The latter have had to be derived from adult germ cells andembryos, and are called embryonal stem (“ES”) cells, embryonal germ(“EG”) cells, and germ cells. Stem cells derived from adult tissue havebeen of limited value because they are immunogenic, have limiteddifferentiation potential, and have limited ability to propagate inculture. ES, EG, and germ cells do not suffer from these disadvantages,but they have a marked propensity to form teratomas in allogeneic hosts,raising due concern for their use in medical treatments. For thisreason, there is pessimism about their utility in clinical applications,despite their advantageously broad differentiation potential. Stem cellsderived from embryos also are subject to ethical controversies that mayimpede their use in treating disease.

Some efforts to find an alternative to ES, EG, and germ cells havefocused on cells derived from adult tissue. While adult stem cells havebeen identified in most tissues of mammals, their differentiationpotential is restricted and considerably more narrow than that of ES,EG, and germ cells. Indeed many such cells can give rise only to one ora few differentiated cell types, and many others are restricted to asingle embryonic lineage.

For instance, hematopoietic stem cells, which can be isolated from bonemarrow, blood, cord blood, fetal liver, and yolk sac, can reinitiatehematopoiesis and generate multiple hematopoietic lineages. Thus, theycan repopulate the erythroid, neutrophil-macrophage, megakaryocyte, andlymphoid hemopoietic cell pools. However, they can differentiate only toform cells of the hematopoietic lineage. They cannot provide cells ofany other lineages.

Neural stem cells were initially identified in the subventricular zoneand the olfactory bulb of fetal brain. Studies in rodents, non-humanprimates, and humans have shown that neural stem cells continue to bepresent in adult brain. These stem cells can proliferate in vivo andcontinuously regenerate at least some neuronal cells in vivo. Whencultured ex vivo, neural stem cells can be induced to proliferate, aswell as to differentiate, into different types of neurons and glialcells. When transplanted into the brain, neural stem cells can engraftand generate neural cells and glial cells. Neural stem cells cannotdifferentiate into cells that are not of neuroectodermal origin.

Mesenchymal stem cells (“MSCs”) originally were derived from theembryonal mesoderm and subsequently have been isolated from adult bonemarrow and other adult tissues. They can be differentiated to formmuscle, bone, cartilage, fat, marrow stroma, and tendon. Mesoderm alsodifferentiates into visceral mesoderm which can give rise to cardiacmuscle, smooth muscle, or blood islands consisting of endothelium andhematopoietic progenitor cells. The differentiation potential of themesenchymal stem cells that have been described thus far is limited tocells of mesenchymal origin, including the best characterizedmesenchymal stem cell (See Pittenger, et al. Science (1999) 284: 143-147and U.S. Pat. No. 5,827,740 (SH2⁺ SH4⁺ CD29⁺ CD44⁺ CD71⁺ CD90⁺ CD106⁺CD120a⁺ CD124⁺ CD14⁻ CD34⁻ CD45⁻)).

For the reasons noted above regarding the limitations, risks, andcontroversies of and relating to ES, EG, and germ cells, a substantialportion of work on the use of stem cells in transplantation therapieshas utilized MSCs. Results of the last few years appear to show thatallografts of MSCs do not engender a HVG immune reaction, which is aresponse invariably seen when other tissue is transplanted betweenallogeneic individuals. Moreover, the results suggest that MSCs weakenlymphocyte immune response, at least in some circumstances.

While these results immediately suggest that MSCs might be useful todecrease HVG and/or GVHD that ordinarily would accompany allogeneictransplantation, the observed immunosuppressive effects of MSCs werehighly dose dependent, and relatively high doses were required toobserve an immunosuppressive effect. In fact, decreased proliferation oflymphocytes in mixed lymphocyte assays in vitro was “marked” only at orabove a 1:10 ratio of MSCs to lymphocytes. Furthermore, the observedinhibitory effect decreased and became unobservable as the dose of MSCsdecreased, and at ratios below 1:100 the presence of MSCs actuallyseemed to stimulate proliferation of the T-cells. The same dose effectsalso were observed in mitogen-stimulated lymphocyte proliferationassays. See, for review, Ryan et al. (2005) “Mesenchymal stem cellsavoid allogeneic rejection,” J. Inflammation 2: 8; Le Blanc (2003)“Immunomodulatory effects of fetal and adult mesenchymal stem cells,”Cytotherapy 5(6): 485-489, and Jorgensen et al. (2003) “Engineeringmesenchymal stem cells for immunotherapy,” Gene Therapy 10: 928-931.Additional results are summarized below.

For example, Bartholomew and co-workers found that baboon MSCs did notstimulate allogeneic lymphocytes to proliferate in vitro and that MSCsreduced proliferation of mitogen-stimulated lymphocytes by more than 50%in mixed lymphocyte assays in vitro. They further showed thatadministration of MSCs in vivo prolonged skin graft survival (relativeto controls). Both the in vitro results and the in vivo results requireda high dose of MSCs: 1:1 ratio with the lymphocytes for the in vitroresults. The amount of MSCs that would be required to approach such aratio in vivo in humans may be too high to achieve, as a practicalmatter. This may limit the utility of MSCs. See Bartholomew et al.(2002): “Mesenchymal stem cells suppress lymphocyte proliferation invitro and prolong skin graft survival in vivo,” Experimental Hematology30: 42-48.

Maitra and co-workers examined the effects of human MSCs on engraftmentof allogeneic human umbilical cord blood cells after co-infusion intosub-lethally irradiated NOD-SCID mice. They found that human MSCspromoted engraftment and did not activate allogeneic T-cells in in vitroproliferation assays. They also found that human MSCs suppressed invitro activation of allogeneic human T-cells by mitogens. The effectswere dose dependent and relatively high ratios were required forsuppression. (Maitra et al. (2004) Bone Marrow Transplantation 33:597-604.)

Recently, Le Blanc and co-workers reported successfully treating onepatient with Grade IV acute GVHD, which usually is fatal, byadministration of “third party haploidentical” MSCs. The patient was a9-year old boy with acute lymphoblastic leukemia, which was in its thirdremission. Initially, the patient was treated with radiation andcyclophophamide and then given blood cells that were identical to hisown cells at the HLA-A, HLA-B, and HLA-DRbetal loci. These had beenobtained from an unrelated female donor. Despite aggressive treatment,including dosing with a variety of immunosuppressants, by 70 days aftertransplant the patient developed Grade IV acute GVHD. He was frequentlyafflicted by invasive bacterial, viral, and fungal infections.

Under these clearly dire circumstances, an alternative blood stem celltransplant was attempted. Haploidentical MSCs were isolated from thepatient's mother and expanded in vitro for three weeks. The cells wereharvested and 2×10⁶ cells per kilogram were administered to the patientintravenously. There were no signs of toxicity associated with the MSCs,nor were there substantial side effects. Many symptoms resolved within afew days after the transplant; but, residual disease was apparent. Afterseveral additional intravenous injections of MSCs using the samemethods, the patient's symptoms and GVHD were fully resolved. Thepatient was still disease free one year after discharge. According tothe authors, in their experience, this patient is unique in survivingGVHD of this severity. The results reported by Le Blanc et al. are bothpromising and inspiring, and should be a spur to developing effectivetherapies that utilize stem cells. Le Blanc et al. (2004) “Treatment ofsevere acute graft-versus-host disease with third party haploidenticalmesenchymal stem cells,” Lancet 363: 1439-41.

Nevertheless, these results, including those of Le Blanc and co-workers,reveal potential shortcomings of MSCs. The cells need to be administeredwith traditional immunosuppressive modalities which then will continueto engender deleterious immune responses. The dosing requirements forMSCs apparently will need to be very high to be effective, which willincur greater cost, more difficulty in administration, greater risk oftoxicity and other harmful side effects, and other disadvantages.

In view of these limitations of current stem cell basedtransplantation-related therapies, there is clearly a strong need forprogenitor cells that can be used for all—or at least most—recipienthosts without necessitating a host-recipient haplotype match. Further,there is a need for cells of greater “specific activity” so that theyare therapeutically effective at lower doses and their administrationdoes not pose the problems associated with the high dosing regimensrequired for beneficial results using MSCs. And, there is a need forcells that have essentially unlimited differentiation potential to formcells that occur in the organism of interest.

Unrestricted Stem Cells

Until recently only ES, EG, and germ cells were thought to have bothunlimited capacity for self-renewal and unrestricted differentiationpotential, i.e., the ability to produce all the different types of cellsand tissues of an organism that occur throughout its embryogenesis,development, adulthood, and senescence. Accordingly, these ES, EG, andgerm cells traditionally have been seen as the most promising stem cellsfor medical uses, such as for applications that involve regeneratinghealthy tissue, and those involving transplantation of cells and/orre-growth of healthy tissue. The embryonal stem (“ES”) cell hasunlimited self-renewal and can differentiate into all tissue types. EScells are derived from the inner cell mass of the blastocyst. Embryonalgerm (“EG”) cells are derived from primordial germ cells of apost-implantation embryo. ES and EG cells have been derived from mouse,and, more recently, from non-human primates and humans. When introducedinto blastocysts, ES cells can contribute to all tissues. A drawback toES cell therapy is that when transplanted in post-natal animals, ES andEG cells generate teratomas.

ES (and EG) cells can be identified by positive staining with antibodiesto SSEA1 (mouse) and SSEA4 (human). At the molecular level, ES and EGcells express a number of transcription factors highly specific forthese undifferentiated cells. These include oct-4 and rex-1. Rex-1expression is controlled by oct-4, which activates downstream expressionof rex-1. Also found are the LIF-R (in mouse) and the transcriptionfactors sox-2 and rox-1. Rox-1 and sox-2 are also expressed in non-EScells. A hallmark of ES cells is the presence of telomerase, whichprovides these cells with an unlimited self-renewal potential in vitro.

Oct-4 (oct-3 in humans) is a transcription factor expressed in thepregastrulation embryo, early cleavage stage embryo, cells of the innercell mass of the blastocyst, and embryonic carcinoma (“EC”) cells(Nichols, J. et al. (1998) Cell 95: 379-91), and is down-regulated whencells are induced to differentiate. The oct-4 gene (oct-3 in humans) istranscribed into at least two splice variants in humans, oct-3A andoct-3B. The oct-3B splice variant is found in many differentiated cellswhereas the oct-3A splice variant (also previously designated oct-3/4)is reported to be specific for the undifferentiated embryonic stem cell.See Shimozaki et al. (2003) Development 130: 2505-12. Expression ofoct-3/4 plays an important role in determining early steps inembryogenesis and differentiation. Oct-3/4, in combination with rox-1,causes transcriptional activation of the Zn-finger protein rex-1, whichis also required for maintaining ES cells in an undifferentiated state(Rosfjord, E. and Rizzino, A. (1997) Biochem Biophys Res Commun 203:1795-802; Ben-Shushan, E. et al. (1998) Mol Cell Biol 18: 1866-78).

In addition, sox-2, expressed in ES/EC cells, but also in other moredifferentiated cells, is needed together with oct-4 to retain theundifferentiated state of ES/EC cells (Uwanogho, D. et al. (1995) MechDev 49: 23-36). Maintenance of murine ES cells and primordial germ cellsrequires the presence of LIF whereas this requirement is not as clearfor human and non-human primate ES cells.

As noted above, ES, EG, and germ cells, despite their seeminglyunlimited differentiation potential, have not been as intense a focus ofattention as they might have been for several reasons. These include,among other things, safety concerns, ethical concerns, logisticalissues, limits on the use of federal funding for research onembryo-derived cells and cell lines (other than a limited number ofspecifically approved human embryonic stem cell lines), economicconsiderations, and political risk factors that have become associatedwith human embryonic stem cell research.

Accordingly, there has been a need for cells that have the self-renewingand differentiation capacity of ES, EG, and germ cells but are notimmunogenic; do not form teratomas when allografted or xenografted to ahost; do not pose other safety issues associated with ES, EG, and germcells; retain the other advantages of ES, EG, and germ cells; are easyto isolate from readily available sources, such as placenta, umbilicalcord, umbilical cord blood, blood, and bone marrow; can be stored safelyfor extended periods; can be obtained easily and without risk tovolunteers, donors or patients, and others giving consent; and do notentail the technical and logistical difficulties involved in obtainingand working with ES, EG, and germ cells.

Recently, a type of cell, called herein multipotent adult progenitorcells (“MAPCs”), has been isolated and characterized. These cellsprovide many of the advantages of ES, EG, and germ cells without many oftheir drawbacks.

SUMMARY OF THE INVENTION

In some of its embodiments, therefore, the invention provides cellsthat: (i) are not embryonic stem cells, not embryonic germ cells, andnot germ cells; (ii) can differentiate into at least one cell type ofeach of at least two of the endodermal, ectodermal, and mesodermalembryonic lineages; (iii) do not provoke a deleterious immune responseupon introduction to a non-syngeneic subject; and (iv) can modulate animmune response upon introduction into a subject. In certain embodimentsin this regard, the invention provides cells that, in addition to theforegoing, are immunosuppressive. Furthermore, various embodiments ofthe invention provide cells in accordance with the foregoing that haveimmunomodulatory properties that are useful for treating, such as topreclude, prevent, ameliorate, lessen, decrease, minimize, eliminate,and/or cure deleterious immune responses and/or processes in a host. Insome embodiments of the invention the cells are used in this regardalone or together with other therapeutic agents and modalities asprimary therapeutic modalities. In some embodiments of the invention thecells are used in an adjunctive therapeutic modality in which they maybe used either as the sole therapeutic agent or together with othertherapeutic agents. In some embodiments of the invention the cells areused, alone or with other therapeutic agents or modalities, both in oneor more primary therapeutic modalities and in one or move adjunctivetherapeutic modalities.

Cells in accordance with the invention are described in greater detailherein and generally are referred to herein as “multipotent adultprogenitor cells” and by the acronyms “MAPC” (singular) and “MAPCs”(plural). It is to be appreciated that these cells are not ES, not EG,and not germ cells, and that they have the capacity to differentiateinto cell types of at least two of the three primitive germ layerlineages (ectoderm, mesoderm, and endoderm), and, in many cases intocells of all three primitive lineages.

MAPCs express telomerase which is considered to be necessary forself-renewal and is thought to be necessary for maintaining anundifferentiated state. Generally they also express oct-3/4. Oct-3/4(oct-3A in humans) is otherwise specific to ES, EG, and germ cells. Itis considered to be a marker of undifferentiated cells that have broaddifferentiation abilities. Oct-3/4 also is generally thought to have arole in maintaining a cell in an undifferentiated state. MAPCs generallyalso express other markers thought to be specific to primitive stemcells. Among these are rex-1, rox-1, and sox-2.

MAPCs are biologically and antigenically distinct from mesenchymal stemcells (“MSCs”). MAPCs have demonstrated differentiation capabilityencompassing the epithelial, endothelial, neural, myogenic,hematopoietic, osteogenic, hepatogenic, chondrogenic, and adipogeniclineages, among others. They are far less restricted in differentiationpotential than MSCs and, therefore, constitute a progenitor cellpopulation that is less committed and less restricted in developmentalpotential than MSCs. They are apparently more primitive progenitor cellsthan MSCs. (Verfaillie, C. M. (2002) Trends Cell Biol. 12(11): 502-8,Jahagirdar, B. N. et al. (2001) Exp Hematol. 29(5): 543-56). Thus, MAPCsare a class of non-ES, non-EG, non-germ cells which emulate the broadbiological plasticity characteristics of ES, EG, and germ cells, whilemaintaining other characteristics that make non-embryonic stem cellsappealing. For example, MAPCs are capable of indefinite culture withoutloss of their differentiation potential. They show efficient, long termengraftment and differentiation along multiple developmental lineages inNOD-SCID mice and do so without evidence of teratoma formation (oftenseen with ES, EG, and germ cells) (Reyes, M. and C. M. Verfaillie (2001)Ann N Y Acad Sci. 938: 231-3; discussion 233-5).

MAPCs were initially isolated from bone marrow but subsequently havebeen established from other tissues, including brain, muscle, and cordblood (Jiang, Y. et al. (2002) Exp Hematol. 30(8): 896-904). MAPCs havebeen prepared from bone marrow tissue by enriching adherent cells inmedia containing low serum (2%) dexamethasone, EGF, PDGF, and otheradditives, and then growing them to high population doublings. At earlyculture points, more heterogeneity is detected in the population. Aroundcell doubling 30, many adherent stromal cells undergo replicativesenescence. The population of cells that continues to expand thereafterbecomes more homogenous and is characterized by cells that have longtelomeres. Methods for obtaining and culturing the cells are describedin greater detail elsewhere herein.

Various embodiments of the invention provide methods for using MAPCs forprecluding, preventing, treating, ameliorating, lessening, decreasing,minimizing, eliminating, and/or curing a disease and/or an adverseimmune response and/or processes in a subject. Certain embodiments ofthe invention provide methods for using the cells by themselves as aprimary therapeutic modality. In some embodiments of the invention thecells are used together with one or more other agents and/or therapeuticmodalities as the primary therapeutic modality. In some embodiments ofthe invention the cells are used as an adjunctive therapeutic modality,that is, as an adjunct to another, primary therapeutic modality. In someembodiments the cells are used as the sole active agent of an adjunctivetherapeutic modality. In others the cells are used as an adjunctivetherapeutic modality together with one or more other agents ortherapeutic modalities. In some embodiments the cells are used both asprimary and as adjunctive therapeutic agents and/or modalities. In bothregards, the cells can be used alone in the primary and/or in theadjunctive modality. They also can be used together with othertherapeutic agents or modalities, in the primary or in the adjunctivemodality or both.

As discussed above, a primary treatment, such as a therapeutic agent,therapy, and/or therapeutic modality, targets (that is, is intended toact on) the primary dysfunction, such as a disease, that is to betreated. An adjunctive treatment, such as a therapy and/or a therapeuticmodality, can be administered in combination with a primary treatment,such as a therapeutic agent, therapy, and/or therapeutic modality, toact on the primary dysfunction, such as a disease, and supplement theeffect of the primary treatment, thereby increasing the overall efficacyof the treatment regimen. An adjunctive treatment, such as an agent,therapy, and/or therapeutic modality, also can be administered to act oncomplications and/or side effects of a primary dysfunction, such as adisease, and/or those caused by a treatment, such as a therapeuticagent, therapy, and/or therapeutic modality. In regard to any of theseuses, one, two, three, or more primary treatments may be used togetherwith one, two, three, or more adjunctive treatments.

In some embodiments MAPCs are administered to a subject prior to onsetof a dysfunction, such as a disease, side effect, and/or deleteriousimmune response. In some embodiments the cells are administered whilethe dysfunction is developing. In some embodiments the cells areadministered after the dysfunction has been established. Cells can beadministered at any stage in the development, persistence, and/orpropagation of the dysfunction or after it recedes.

As discussed above, embodiments of the invention provide cells andmethods for primary or adjunctive therapy. In certain embodiments of theinvention, the cells are administered to an allogeneic subject. In someembodiments they are autologous to the subject. In some embodiments theyare syngeneic to the subject. In some embodiments the cells arexenogeneic to a subject. Whether allogeneic, autologous, syngeneic, orxenogeneic, in various embodiments of the invention the MAPCs are weaklyimmunogenic or are non-immunogenic in the subject. In some embodimentsthe MAPCs have sufficiently low immunogenicity or are non-immunogenicand are sufficiently free of deleterious immune responses in general,that when administered to allogeneic subjects they can be used as“universal” donor cells without tissue typing and matching. Inaccordance with various embodiments of the invention the MAPCs can alsobe stored and maintained in cell banks, and thus can be kept availablefor use when needed.

In all of these regards and others, embodiments of the invention provideMAPCs from mammals, including in one embodiment humans, and in otherembodiments non-human primates, rats and mice, and dogs, pigs, goats,sheep, horses, and cows. MAPCs prepared from mammals as described abovecan be used in all of the methods and other aspects of the inventiondescribed herein.

MAPCs in accordance with various embodiments of the invention can beisolated from a variety of compartments and tissues of such mammals,including but not limited to, bone marrow, blood, spleen, liver, muscle,brain, and others discussed below. MAPCs in some embodiments arecultured before use.

In some embodiments MAPCs are genetically engineered, such as to improvetheir immunomodulatory properties. In some embodiments geneticallyengineered MAPCs are produced by in vitro culture. In some embodimentsgenetically engineered MAPCs are produced from a transgenic organism.

In various embodiments the MAPCs are administered to a subject by anyroute for effective delivery of cell therapeutics. In some embodimentsthe cells are administered by injection, including local and/or systemicinjection. In certain embodiments the cells are administered withinand/or in proximity to the site of the dysfunction they are intended totreat. In some embodiments, the cells are administered by injection at alocation not in proximity to the site of the dysfunction. In someembodiments the cells are administered by systemic injection, such asintravenous injection.

In some embodiments, MAPCs are administered one time, two times, threetimes, or more than three times until a desired therapeutic effect isachieved or administration no longer appears to be likely to provide abenefit to the subject. In some embodiments MAPCs are administeredcontinuously for a period of time, such as by intravenous drip.Administration of MAPCs may be for a short period of time, for days, forweeks, for months, for years, or for longer periods of time.

The following numbered paragraphs describe a few illustrativeembodiments of the invention that exemplify some of its aspects andfeatures. They are not exhaustive in illustrating its many aspects andembodiments, and thus are not in any way limitative of the invention.Many other aspects, features, and embodiments of the invention aredescribed herein. Many other aspects and embodiments will be readilyapparent to those skilled in the art upon reading the application andgiving it due consideration in the full light of the prior art andknowledge in the field.

The numbered paragraphs below are self-referential. The phrase“according to any of the foregoing or the following” refers to all ofthe preceding and all of the following numbered paragraphs and theircontents. All phrases of the form “according to #” are direct referencesto that numbered paragraph, e.g., “according to 46.” means according toparagraph 46. in this collection of numbered paragraphs. Allcross-references are combinatorial, except for redundancies andinconsistencies of scope. The cross-references are used explicitly toprovide a concise description showing the inclusion of the variouscombinations of subject matter with one another.

1. A method of treating an immune dysfunction in a subject, comprising:administering to a subject likely to suffer, suffering, or who hassuffered from an immune dysfunction, by an effective route and in aneffective amount to treat the immune dysfunction, cells (MAPCs) that:are not embryonic stem cells, embryonic germ cells, or germ cells; candifferentiate into at least one cell type of each of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages; do notprovoke a deleterious immune response in the subject; and are effectiveto treat the immune dysfunction.

2. A method of adjunctive treatment of a subject, comprising:administering to a subject likely to suffer, suffering, or who hassuffered from an immune dysfunction, by an effective route and in aneffective amount to treat the immune dysfunction, cells (MAPCs) that:are not embryonic stem cells, embryonic germ cells, or germ cells; candifferentiate into at least one cell type of each of at least two of theendodermal, ectodermal, and mesodermal embryonic lineages; do notprovoke a deleterious immune response in the subject; and are effectiveto treat the immune dysfunction, wherein the cells are administeredadjunctively to one or more other treatments administered to the subjectto treat the same thing, to treat something different, or both.

3. A method according to any of the foregoing or the following, whereinsaid cells can differentiate into at least one cell type of each of theendodermal, ectodermal, and mesodermal embryonic lineages.

4. A method according to any of the foregoing or the following, whereinsaid cells express telomerase.

5. A method according to any of the foregoing or the following, whereinsaid cells are positive for oct-3/4.

6. A method according to any of the foregoing or the following, whereinsaid cells have undergone at least 10 to 40 cell doublings in cultureprior to their administration to the subject.

7. A method according to any of the foregoing or the following, whereinsaid cells are mammalian cells.

8. A method according to any of the foregoing or the following, whereinsaid cells are human, horse, cow, goat, sheep, pig, rat, or mouse cells.

9. A method according to any of the foregoing or the following, whereinsaid cells are human, rat, or mouse cells.

10. A method according to any of the foregoing or the following, whereinsaid cells are human cells.

11. A method according to any of the foregoing or the following, whereinsaid cells are derived from cells isolated from any of placental tissue,umbilical cord tissue, umbilical cord blood, bone marrow, blood, spleentissue, thymus tissue, spinal cord tissue, adipose tissue, and livertissue.

12. A method according to any of the foregoing or the following, whereinsaid cells are derived from cells isolated from any of placental tissue,umbilical cord tissue, umbilical cord blood, bone marrow, blood, andspleen tissue.

13. A method according to any of the foregoing or the following, whereinsaid cells are derived from cells isolated from any of placental tissue,umbilical cord tissue, umbilical cord blood, bone marrow, or blood.

14. A method according to any of the foregoing or the following, whereinsaid cells are derived from cells isolated from any one or more of bonemarrow or blood.

15. A method according to any of the foregoing or the following, whereinsaid cells are allogeneic to the subject.

16. A method according to any of the foregoing or the following, whereinsaid cells are xenogeneic to the subject.

17. A method according to any of the foregoing or the following, whereinsaid cells are autologous to the subject.

18. A method according to any of the foregoing or the following whereinthe subject is a mammal.

19. A method according to any of the foregoing or the following whereinthe subject is a mammalian pet animal, a mammalian livestock animal, amammalian research animal, or a non-human primate.

20. A method according to any of the foregoing or the following, whereinthe subject is a human.

21. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in one or more dosescomprising 10⁴ to 10⁸ of said cells per kilogram of the subject's mass.

22. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in one or more dosescomprising 10⁵ to 10⁷ of said cells per kilogram of the subject's mass.

23. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in one or more dosescomprising 5×10⁶ to 5×10⁷ of said cells per kilogram of the subject'smass.

24. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in one or more dosescomprising 2×10⁷ to 4×10⁷ of said cells per kilogram of the subject'smass.

25. A method according to any of the foregoing or the following, whereinin addition to said cells, one or more factors are administered to saidsubject.

26. A method according to any of the foregoing or the following, whereinin addition to said cells, one or more growth factors, differentiationfactors, signaling factors, and/or factors that increase homing areadministered to said subject.

27. A method according to any of the foregoing or the following, whereinin addition to said cells, one or more cytokines are administered tosaid subject.

28. A method according to any of the foregoing or the following, whereinsaid cells are administered to a subject adjunctively to anothertreatment that is administered before, at the same time as, or aftersaid cells are administered.

29. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively toadministration to the subject of one or more immunosuppressive agents.

30. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a transplant, wherein said cells are administeredadjunctively thereto.

31. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a transplant of a kidney, heart, lung, liver, or otherorgan, wherein said cells are administered adjunctively thereto.

32. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a transplant of bone marrow, vein, artery, muscle, or othertissue, wherein said cells are administered adjunctively thereto.

33. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a transplant of blood cells, islet cells, or other tissueor organ regenerating cells, wherein said cells are administeredadjunctively thereto.

34. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a blood cell transplant, wherein said cells areadministered adjunctively thereto.

35. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject is to receive orhas received a bone marrow transplant, wherein said cells areadministered adjunctively thereto.

36. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject has been, will be,or is being treated with one or more immunosuppressive agents, whereinsaid cells are administered adjunctively thereto.

37. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject has been, will be,or is being treated with one or more of a corticosteroid, cyclosporin A,a cyclosporin-like immunosuppressive agent, cyclophosphamide,antithymocyte globulin, azathioprine, rapamycin, FK-506, and amacrolide-like immunosuppressive agent other than FK-506, rapamycin, andan immunosuppressive monoclonal antibody agent (i.e., animmunosuppressive that is an immunosuppressive monoclonal antibody or isan agent comprising a monoclonal antibody, in whole or in one or moreparts, such as a chimeric protein comprising an Fc or a Ag binding siteof a monoclonal antibody), wherein said cells are administeredadjunctively thereto.

38. A method according to any of the foregoing or the following, whereinin addition to treatment with said cells, the subject has been, will be,or is being treated with one or more of a corticosteroid, cyclosporin A,azathioprine, rapamycin, cyclophosphamide, FK-506, or animmunosuppressive monoclonal antibody agent, wherein said cells areadministered adjunctively thereto.

39. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively toadministration to the subject of one or more antibiotic agents.

40. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively toadministration to the subject of one or more anti-fungal agents.

41. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively toadministration to the subject of one or more anti-viral agents.

42. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively to theadministration to the subject of any combination of two or more of anyimmunosuppressive agents and/or antibiotic agents and/or anti-fungalagents and/or anti-viral agents.

43. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject adjunctively to a transplanttherapy to treat a host versus graft response in the subject that isimpairing or might impair the therapeutic efficacy of the transplantand/or is or might result in transplant rejection.

44. A method according to any of the foregoing or the following, whereinsaid cells are administered to a subject having a weakened immunesystem, such as one or more of a compromised immune system and/or anablated immune system.

45. A method according to any of the foregoing or the following, whereinsaid cells are administered to a subject adjunctively to radiationtherapy or chemotherapy or a combination of radiation and chemotherapythat either have been, are being, or will be administered to thesubject.

46. A method according to any of the foregoing or the following, whereinsaid cells are administered to a subject adjunctively to an on-goingregimen of radiation therapy or chemotherapy or a combination ofradiation and chemotherapy.

47. A method according to any of the foregoing or the following, whereinthe immune system of the subject has been weakened, compromised, and/oror ablated by radiation therapy, chemotherapy, or a combination ofradiation and chemotherapy.

48. A method according to any of the foregoing or the following, whereinthe subject is the recipient of a non-syngeneic blood cell or bonemarrow transplant, the immune system of the subject has been weakened orablated by radiation therapy, chemotherapy, or a combination ofradiation and chemotherapy, and the subject is at risk to develop or hasdeveloped graft versus host disease.

49. A method according to any of the foregoing or the following, whereinthe subject is the recipient of a non-syngeneic blood cell or bonemarrow transplant, the immune system of the subject has been weakened orablated by radiation therapy, by chemotherapy, or by a combination ofradiation therapy and chemotherapy, and immunosuppressive drugs arebeing administered to the subject, wherein further the subject is atrisk to develop or has developed graft versus host disease and saidcells are administered to said subject to treat graft versus hostdisease adjunctively to one or more of the other treatments (that is:the transplant, the radiation therapy, the chemotherapy, and/or theimmunosuppressive drugs).

50. A method according to any of the foregoing or the following, whereinthe subject will be or is the recipient of a non-syngeneic transplantand is at risk for or has developed a host versus graft response,wherein said cells are administered to treat the host versus graftresponse.

51. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a neoplasm and saidcells are administered adjunctive to a treatment thereof.

52. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a neoplasm of blood orbone marrow cells and said cells are administered adjunctive to atreatment thereof.

53. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a benign neoplasm ofbone marrow cells, a myeloproliferative disorder, a myelodysplasticsyndrome, or an acute leukemia and said cells are administeredadjunctive to a treatment thereof.

54. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a benign neoplasm ofbone marrow cells and said cells are administered adjunctive to atreatment thereof.

55. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a myeloproliferativedisorder and said cells are administered adjunctive to a treatmentthereof.

56. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more of chronicmyelocytic leukemia (“CML”) (also called chronic granulocytic leukemia(“CGL”)), agnogenic myelofibrosis, essential thrombocythemia,polycythemia vera, or other myeloproliferative disorder and said cellsare administered adjunctive to a treatment thereof.

57. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a myelodysplasticsyndrome and said cells are administered adjunctive to a treatmentthereof.

58. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from an acute leukemia andsaid cells are administered adjunctive to a treatment thereof.

59. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more of acutemultiple myeloma, myeloblastic leukemia, chronic myelocytic leukemia(“CML”), acute promyelocytic leukemia, pre-B acute lymphoblasticleukemia, chronic lymphocytic leukemia (“CLL”), B-cell lymphoma, hairycell leukemia, myeloma, T-acute lymphoblastic leukemia, peripheralT-cell lymphoma, other lymphoid leukemias, other lymphomas, or otheracute leukemia and said cells are administered adjunctive to a treatmentthereof.

60. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from an anemia or other blooddisorder and said cells are administered adjunctive to a treatmentthereof.

61. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from hemoglobinopathies,thalassemia, bone marrow failure syndrome, sickle cell anemia, aplasticanemia, Fanconi's anemia, or an immune hemolytic anemia and said cellsare administered adjunctive to a treatment thereof.

62. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more ofrefractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, chronic myelomonocytic leukemia, or othermyelodysplastic syndrome and said cells are administered adjunctive to atreatment thereof.

63. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from Fanconi's anemia andsaid cells are administered adjunctive to a treatment thereof.

64. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from an immune dysfunctionand said cells are administered adjunctive to a treatment thereof.

65. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from a congenital immunedeficiency and said cells are administered adjunctive to a treatmentthereof.

66. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from an autoimmunedysfunction, disorder, or disease and said cells are administeredadjunctive to a treatment thereof.

67. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more of thefollowing autoimmune dysfunctions: Crohn's disease, Guillain-Barrésyndrome, lupus erythematosus (also called “SLE” and systemic lupuserythematosus), multiple sclerosis, myasthenia gravis, optic neuritis,psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease,Ord's thyroiditis, diabetes mellitus (type 1), Reiter's syndrome,autoimmune hepatitis, primary biliary cirrhosis, antiphospholipidantibody syndrome (“APS”), opsoclonus-myoclonus syndrome (“OMS”),temporal arteritis, acute disseminated encephalomyelitis (“ADEM” and“ADE”), Goodpasture's, syndrome, Wegener's granulomatosis, celiacdisease, pemphigus, polyarthritis, and warm autoimmune hemolytic anemiaand said cells are administered adjunctive to a treatment thereof.

68. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more of thefollowing autoimmune dysfunctions: Crohn's disease, lupus erythematosus(also called “SLE” and systemic lupus erythematosus), multiplesclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, Graves'disease, Hashimoto's disease, diabetes mellitus (type 1), Reiter'ssyndrome, primary biliary cirrhosis, celiac disease, polyarthritis, andwarm autoimmune hemolytic anemia and said cells are administeredadjunctive to a treatment thereof.

69. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from one or more of thefollowing diseases thought to have an autoimmune component:endometriosis, interstitial cystitis, neuromyotonia, scleroderma,progressive systemic scleroderma, vitiligo, vulvodynia, Chagas' disease,sarcoidosis, chronic fatigue syndrome, and dysautonomia and said cellsare administered adjunctive to a treatment thereof.

70. A method according to any of the foregoing or the following, whereinthe subject is at risk for or is suffering from an inflammatory diseaseand said cells are administered adjunctive to a treatment thereof.

71. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or moreother pharmaceutically active agents.

72. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or moreother immunosuppressive agents.

73. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or more of acorticosteroid, cyclosporin A, a cyclosporin-like immunosuppressiveagent, cyclophosphamide, antithymocyte globulin, azathioprine,rapamycin, FK-506, and a macrolide-like immunosuppressive agent otherthan FK-506, rapamycin, and an immunosuppressive monoclonal antibodyagent.

74. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or more of acorticosteroid, cyclosporin A, azathioprine, cyclophosphamide,rapamycin, FK-506, and an immunosuppressive monoclonal antibody agent.

75. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or moreantibiotic agents.

76. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or moreantifungal agents.

77. A method according to any of the foregoing or the following, whereinsaid cells are administered in a formulation comprising one or moreantiviral agents.

78. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject by a parenteral route.

79. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject by any one or more of thefollowing parenteral routes: intravenous, intraarterial, intracardiac,intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial,intracutaneous, intradermal, subcutaneous, and intramuscular injection.

80. A method according to any of the foregoing or the following, whereinsaid cells are administered by any one or more of the followingparenteral routes: intravenous, intraarterial, intracutaneous,intradermal, subcutaneous, and intramuscular injection.

81. A method according to any of the foregoing or the following, whereinsaid cells are administered by any one or more of the followingparenteral routes: intravenous, intraarterial, intracutaneous,subcutaneous, and intramuscular injection.

82. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject through a hypodermic needleby a syringe.

83. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject through a catheter.

84. A method according to any of the foregoing or the following, whereinsaid cells are administered by surgical implantation.

85. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject by implantation using anarthroscopic procedure.

86. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in or on a support.

87. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in an encapsulated form.

88. A method according to any of the foregoing or the following, whereinsaid cells are formulated suitably for administration by any one or moreof the following routes: oral, rectal, epicutaneous, ocular, nasal, andpulmonary.

89. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in one dose.

90. A method according to any of the foregoing or the following, whereinsaid cells are administered to the subject in a series of two or moredoses in succession.

91. A method according to any of the foregoing or the following, whereinsaid cells are administered in a single dose, in two doses, or in morethan two doses, wherein the doses are the same or different, and theyare administered with equal or with unequal intervals between them.

92. A method according to any of the foregoing or the following, whereinsaid cells are administered over a period of less than one day to oneweek, one week to one month, one month to one year, one year to twoyears, or longer than two years.

93. A method of treatment of an immune dysfunction in a subject,comprising administering to a subject suffering from an immunedysfunction, by a route and in an amount effective for treating theimmune dysfunction in the subject, cells that: are not embryonic stemcells, embryonic germ cells, or germ cells; can differentiate into atleast one cell type of each of at least two of the endodermal,ectodermal, and mesodermal embryonic lineages; do not provoke adeleterious immune response in the subject; and are effective to treatthe immune dysfunction in the subject.

94. A method of adjunctive treatment of an immune dysfunction in asubject, comprising administering to a subject suffering from an immunedysfunction, by a route and in an amount effective for treating theimmune dysfunction in the subject, cells that: are not embryonic stemcells, embryonic germ cells, or germ cells; can differentiate into atleast one cell type of each of at least two of the endodermal,ectodermal, and mesodermal embryonic lineages; do not provoke adeleterious immune response in the subject; and are effective to treatthe immune dysfunction in the subject, wherein the cells areadministered to the subject adjunctively to one or more other treatmentsthat are being administered to the subject to treat the same immunedysfunction, to treat one or more other dysfunctions, or both.

Other aspects of the invention are described in or are obvious from thefollowing disclosure, and are within the ambit of the invention.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 is a schematic representation of the transcriptional profilingstudies that were performed to generate (identify) gene and surfacereceptor-based markers that distinguish between MAPCs of the inventionand other stem and progenitor cells that are more lineage committed. Theexperiments have resulted in a panel of 75 markers having 10-folddifferent expression between MSC cultures and the MAPCs.

FIG. 2 is a set of graphs showing the tri-lineage differentiation ofGFP-labeled rat MAPCs. The results show that MAPCs can differentiateinto cells of all three embryonic lineages. As further described below,for endothelial differentiation, MAPCs were cultured onfibronectin-coated plates in the presence of vascular endothelial growthfactor B (VEGF-B). For hepatocyte differentiation, cells were grown onmatrigel-coated plates and treated with fibroblast growth factor-4(FGF-4) and hepatocyte growth factor (HGF). Neuronal differentiation wasinduced by sequential treatment with basic-FGF (bFGF), with both FGF-8and Sonic Hedgehog (SHH), and with brain-derived neurotrophic factor(BDNF). After two weeks, mRNA was extracted from cells and applied toqPCR analysis using primers specific for detection of various lineagemarkers. In all assays, cells cultured in the absence oflineage-inducing cytokines served as controls. The expression levels oflineage markers were first normalized to the expression level of aninternal control gene (GAPDH) which is unaffected duringdifferentiation. Differentiation success was then assessed bycalculation of the relative expression in the differentiated or thecontrol cells compared to the levels in the parental rat line, using anincrease of more than 5-fold in the relative expression as a cut-off forsuccessful differentiation. Differentiated rat MAPCs displayedsignificant expression of the endothelial markers, von Willebrandfactor, and PECAM-1 (top panel); the hepatic markers albumin,cytokeratin-18, and HNF-1a (middle panel); and the neuronal/astrocytemarkers GFAP, nestin, and NF-200 (bottom panel).

FIG. 3 is a pair of bar charts showing the low immunogenicity (toppanel) and immunosuppressivity (bottom panel) of MAPCs in mixedlymphocyte reactions (MLR), as described further below. In the toppanel: B+B=donor B+donor B; B+A=donor B+donor A; B+K=donor B+donor K;B+R=donor B+donor R; B+T=donor B+donor T; donor B+PHA; B+BMPC=donorB+MAPC. The same result was achieved with twelve different donors. Inthe bottom panel: donor W+donor W; donor W+donor A; donor W+donor T;donor W+MSC; donor W+MAPC (17); donor W+PHA; donor W+donor A+MSC; donorW+donor A+MAPC (17); donor W+donor T+MSC; donor W+donor T+MAPC (17);donor W+donor P+MSC; donor W+donor P+MAPC (17). PHA isphytohemagglutinin (positive control for T-cell activation).

FIG. 4 is a chart showing that MAPCs can suppress the proliferation ofConA stimulated T-cells as described in Example 6. The caption “LN Only”designates the results for control reactions omitting MAPCs. The numbersfor MAPCs indicate how many cells were used in the assays.

FIG. 5A is a chart showing the immunosuppressive effects of Lewis MAPCsin mixed lymphocyte reactions, as described in Example 7. The box inFIG. 5A enumerates the number of MAPCs in each reaction. In the box, Rdesignates responder cells and S designates stimulator cells(splenocytes from irradiated DA rats).

FIG. 5B is a chart showing the immunosuppressive effects ofSprague-Dawley MAPCs in mixed lymphocyte reactions, as described inExample 7. Captions and abbreviations are the same as in FIG. 5A.

FIG. 6 is a graph showing that infusion of MAPCs does not adverselyaffect the health of recipients as determined by their respiratory rate.The graph is further described in Example 8.

FIG. 7 is a bar chart that depicts the results of an experimentdemonstrating the ability of MAPCs to suppress an on-going immuneresponse. The chart shows that MAPCs are strongly immunosuppressive inMLRs, both when they are added at the same time as the T-cell activator(stimulator) (Day 0, left side of chart), and when they are added 3 daysafter addition of the T-cell activator (stimulator) (Day 3, right sideof chart). Details of the experiments are further described in Example10.

DEFINITIONS

As used herein, certain terms have the meanings set out below.

“A” or “an” means one or more; at least one.

“Adjunctive” means jointly, together with, in addition to, inconjunction with, and the like.

“Co-administer” can include simultaneous or sequential administration oftwo or more agents.

“Cytokines” refer to cellular factors that induce or enhance cellularmovement, such as homing of MAPCs or other stem cells, progenitor cells,or differentiated cells. Cytokines may also stimulate such cells todivide.

“Deleterious” means, as used herein, harmful. By way of illustration,“deleterious immune response” means, as used herein, a harmful immuneresponse, such as those that are lacking or are too weak, those that aretoo strong, and/or those that are misdirected. Among deleterious immuneresponses are the harmful immune responses that occur in immunediseases. Examples include the lack of immune responses inimmunodeficiency diseases, and the exaggerated and/or misdirected immuneresponses in autoimmune diseases. Also among deleterious immuneresponses are immune responses that interfere with medical treatment,including otherwise normal immune responses. Examples include immuneresponses involved in rejecting transplants and grafts, and the responseof immunocompetent cells in transplants and grafts that cause graftversus host disease.

“Differentiation factors” refer to cellular factors, such as growthfactors, that induce lineage commitment.

“Dysfunction” means, as used herein, a disorder, disease, or deleteriouseffect of an otherwise normal process. By way of illustration, an immunedysfunction includes immune diseases, such as autoimmune diseases andimmune deficiencies. It also includes immune responses that interferewith medical treatment, including otherwise normal immune responses thatinterfere with medical treatment. Examples of such dysfunctions includeimmune responses involved in rejecting transplants and grafts, and theresponse of immunocompetent cells in transplants and grafts that causegraft versus host disease.

“EC cells” refers to embryonic carcinoma cells.

“Effective amount” generally means an amount which provides the desiredlocal or systemic effect. For example, an effective amount is an amountsufficient to effectuate a beneficial or desired clinical result. Theeffective amounts can be provided all at once in a single administrationor in fractional amounts that provide the effective amount in severaladministrations. For instance, an effective amount of MAPCs could beadministered in one or more administrations and could include anypreselected amount of cells. The precise determination of what would beconsidered an effective amount may be based on factors individual toeach subject, including their size, age, injury, and/or disease orinjury being treated, and amount of time since the injury occurred orthe disease began. One skilled in the art will be able to determine theeffective amount for a given subject based on these considerations whichare routine in the art. Thus, for instance, the skilled artisan in thisart, such as a physician, based on the known properties of MAPCs asdisclosed herein and in the art, together with a consideration of theforegoing factors, will be able to determine the effective amount ofMAPCs for a given subject. As used herein, “effective dose” means thesame as “effective amount.”

“EG cells” refers to embryonal germ cells.

“Engraft” refers to the process of cellular contact and incorporationinto an existing tissue of interest in vivo.

“Enriched population” means a relative increase in numbers of MAPCsrelative to other cells or constituents in an initial population, suchas an increase in numbers of MAPCs relative to one or more non-MAPC celltypes in culture, such as primary culture, or in vivo.

“ES cells” refers to embryonal stem cells.

“Expansion” refers to the propagation of a cell or cells withoutdifferentiation.

“Fanconi's anemia” as used herein means the same as Fanconi anemia, aninherited disease.

“GVHD” refers to graft versus host disease, which means processes thatoccur primarily in an immunocompromised host when it is recognized asnon-self by immunocompetent cells of a graft.

“HVG” refers to host versus graft response, which means processes whichoccur when a host rejects a graft. Typically, HVG is triggered when agraft is recognized as foreign (non-self) by immunocompetent cells ofthe host.

“Isolated” refers to a cell or cells which are not associated with oneor more cells or one or more cellular components that are associatedwith the cell or cells in vivo.

“MAPC” is an acronym for “multipotent adult progenitor cell.” It refersto a non-ES, non-EG, non-germ cell that can give rise to cell lineagesof more than one germ layer, such as all three germ layers (i.e.,endoderm, mesoderm, and ectoderm). MAPCs also may express telomerase.They may be positive for oct-3/4 (e.g., human oct-3A). They also mayexpress rex-1 and rox-1. Further, they may express sox-2, SSEA-4, and/ornanog. The term “adult” in MAPC is not restrictive. It only denotes thatthese cells are not ES, EG, or germ cells. Typically, as used herein,MAPC is singular and MAPCs is plural. MAPCs also have been referred toas multipotent adult stem cells (MASCs) and as multipotent progenitorcells (MPCs).

MAPCs may constitutively express oct-3/4 and high levels of telomerase(Jiang, Y. et al. (2002) Nature 418(6893): 41; Exp Hematol. 30(8): 896.MAPCs derived from human, mouse, rat, or other mammals appear to be theonly normal, non-malignant, somatic cells (non-germ cells) known to dateto express very high levels of telomerase even in late passage cells.The telomeres are extended in many MAPCs, and MAPCs are karyotypicallynormal. MAPCs are self-renewing stem cells that can be cultured throughmany passages without differentiation. They also can be stimulated todifferentiate into a wide variety of cells. In addition, MAPCs injectedinto a mammal can migrate to and assimilate within multiple organs. Assuch, they have utility in the repopulation of organs, either in aself-renewing state or in a differentiated state compatible with theorgan of interest. They have the capacity to replace cell types thatcould have been damaged, died, or otherwise might have an abnormalfunction because of genetic or acquired disease. They may contribute tothe preservation of healthy cells or production of new cells in atissue.

“MASC,” see MAPC.

“MNC” refers to mononuclear cells.

“Modality” means a type, approach, avenue, or method, such as, atherapeutic modality; i.e., a type of therapy.

“MPC,” see MAPC.

“MSC” is an acronym for mesenchymal stem cells.

“Multipotent” with respect to MAPCs, refers to the ability to give riseto cell lineages of more than one germ layer, such as all threeprimitive germ layers (i.e., endoderm, mesoderm, and ectoderm) upondifferentiation.

“Persistence” refers to the ability of cells to resist rejection andremain and/or increase in number over time (e.g., days, weeks, months,or years) in vivo.

“Progenitor” as used in multipotent adult progenitor cells (MAPCs)indicates that these cells can give rise to other cells such as furtherdifferentiated cells. The term is not limitative and does not limitthese cells to a particular lineage.

“Self-renewal” refers to the ability to produce replicate daughter stemcells having differentiation potential that is identical to those fromwhich they arose. A similar term used in this context is“proliferation.”

A “subject” is a vertebrate, such as a mammal, such as a human. Mammalsinclude, but are not limited to, humans, farm animals, sport animals,and pets. Subjects in need of treatment by methods of the presentinvention include those suffering from a disorder, dysfunction, ordisease (such as an immune deficiency or dysfunction, such as HVG andGVHD), or a side effect of the same, or a treatment thereof, that canbenefit from administration of MAPCs either as a primary or anadjunctive treatment.

“Transplant” as used herein means to introduce into a subject, cells,tissues, or organs. The transplant can be derived from the subject, fromculture, or from a non-subject source.

“Treat,” “treating,” or “treatment” includes treating, preventing,ameliorating, inhibiting, or curing a deficiency, dysfunction, disease,or other process resulting in a deleterious effect, such as an immunesystem deficiency, dysfunction, disease, or other process thatdeleteriously affects immune system functions or properties or thatinterferes with a therapy.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The cells described herein (MAPCs) have the ability to regenerate atleast two and often all three primitive germ layers (endodermal,mesodermal, and ectodermal) in vitro and in vivo. In this context theyare equivalent to embryonal stem cells and distinct from mesenchymalstem cells. The biological potency of MAPCs has been proven in variousanimal models, including mouse, rat, and xenogeneic engraftment of humanstem cells in rats and NOD/SCID mice (Reyes, M. and C. M. Verfaillie(2001) Ann N Y Acad Sci. 938: 231-3, discussion pp. 233-5; Jiang, Y. etal. (2002) Exp Hematol. 30(8): 896-904).

The clonal potency of MAPCs has been conclusively demonstrated byinjecting single genetically marked MAPCs into mouse blastocysts,implanting the blastocysts, and allowing the embryos to develop to term(Jiang, Y. et al. (2002) Nature 418(6893): 41-9). Post-natal markeranalysis showed that all of the animals were highly chimeric and thatthe single injected MAPCs proliferated to form differentiated cells ofall tissues and organs. No abnormalities or organ dysfunction wereobserved in any of the animals.

Accordingly, MAPCs are very promising for treating disease by celltransplantation techniques, such as for tissue and organ regeneration,both when used alone and when used in combination with other treatments.Among the potential obstacles to realizing the promise of MAPCs fortreating diseases, and for tissue and organ regeneration, are theadverse immune reactions that typically complicate or prevent success intransplantation therapies, such as blood and bone marrow transplantationtherapies and solid organ transplantation. Prominent among these immunecomplications are graft rejection by a host's immune system (referred toherein as host versus graft response and as “HVG”) and systemic damageto an immunocompromised host that results when immunocompetent cells ina graft are activated by contact with non-self components of the host(referred to herein as graft versus host disease and as “GVHD”).

It has been found (as described in greater detail elsewhere herein) thatMAPCs do not provoke an immune response in allogeneic hosts. Thus,transplantation of MAPCs to an allogeneic host should not engenderallogeneic graft rejection (i.e., HVG).

Furthermore, it has also been found that allogeneic MAPCs can beadministered to a host at high concentration without deleterious effectson respiration, suggesting that undue clumping and/or deposition in thelungs does not occur.

In addition, it has been found (as described in greater detail elsewhereherein) that MAPCs can modulate immune responses. In particular in thisregard, it has been found that MAPCs can suppress immune responses,including but not limited to immune responses involved in, for example,HVG and GVHD, to name just two. In an even more detailed particular inthis regard, it has been found that MAPCs can suppress proliferation ofT-cells, even in the presence of potent T-cell stimulators, such asConcanavalin A and allogeneic stimulator cells.

Moreover, it has been found that even relatively small amounts of MAPCscan suppress these responses. Indeed, only 3% MAPCs in mixed lymphocytereactions is sufficient to reduce T-cell response to potent stimulatorsby 50% in vitro.

Accordingly, in certain aspects of the invention in this regard, certainof the embodiments provide compositions and methods and the like fortreating, ameliorating, and/or curing or eliminating, adverse immunereactions, such as those that occur in transplantation therapies.

The low immunogenicity of allogeneic MAPCs, their ability to suppressadverse immune responses, and their high specific activity makes themparticularly valuable for adjunctive therapies in the treatment ofdiseases with an adverse immune component. Among such diseases areautoimmune diseases in which, typically, dysfunction of the subject'sown immune system causes disease. MAPCs also are useful asimmunosuppressive adjunctive therapeutics for treating adverse immuneresponses that occur in transplantation therapy. Examples include HVG inimmunocompetent hosts and GVHD in immunocompromised hosts. MAPCs furthercan be useful in adjunctive immunosuppressive therapy in the treatmentof a variety of neoplasms, anemias and blood disorders, and in thetreatment of certain inflammatory diseases. Diseases in this regard arediscussed in greater detail below.

Using the methods described herein for MAPC isolation, characterization,and expansion, together with the disclosure herein on immune-suppressingproperties of MAPCs, MAPCs can be used to prevent, suppress, or diminishimmune disorders, dysfunctions, or diseases, including, for example,adverse immune reactions, such as those that result from othertherapies, including those that complicate transplantation therapies,such as HVG and GVHD. Such disorders, dysfunctions, and diseases alsoinclude congenital immune disorders and autoimmune diseases, amongothers.

MAPCs are useful in these regards and others both as primary andadjunctive therapeutic agents and modalities. MAPCs can be usedtherapeutically alone or together with other agents. MAPCs can beadministered before, during, and/or after such agents. Likewise, whetherused alone or with other agents, MAPCs can be administered before,during, and/or after a transplant. If administered during transplant,MAPCs can be administered together with the transplant material orseparately. If separately administered, the MAPCs can be administeredsequentially or simultaneously with the transplant. Furthermore, MAPCsmay be administered in advance of the transplant and/or after thetransplant.

Other agents that can be used in conjunction with MAPCs, intransplantation therapies in particular, include immunomodulatoryagents. A variety of such agents are described elsewhere herein. Incertain embodiments of the invention, the immunomodulatory agents areimmunosuppressive agents, such as those described elsewhere herein.Among such agents are corticosteroids, cyclosporin A, cyclosporin-likeimmunosuppressive compounds, azathioprine, cyclophosphamide, andmethotrexate.

MAPCs can be administered to hosts by a variety of methods as discussedelsewhere herein. In certain embodiments the MAPCs are administered byinjection, such as by intravenous injection. In some embodiments MAPCsare encapsulated for administration. In some embodiments the MAPCs areadministered in situ. Examples include in situ administration of MAPCsin solid organ transplantation and in organ repair. These and otherforms of administration are discussed below.

In some embodiments of the invention, MAPCs are administered in dosesmeasured by the ratio of MAPCs (cells) to body mass (weight).Alternatively, MAPCs can be administered in doses of a fixed number ofcells. Dosing, routes of administration, formulations, and the like arediscussed in greater detail elsewhere herein.

Mechanisms of Action

Without being limited to any one or more explanatory mechanisms for theimmunomodulatory and other properties, activities, and effects of MAPCs,it is worth nothing that they can modulate immune responses through avariety of modalities. For instance, MAPCs can have direct effects on agraft or host. Such direct effects are primarily a matter of directcontact between MAPCs and cells of the host or graft. The contact may bewith structural members of the cells or with constituents in theirimmediate environment. Such direct mechanisms may involve directcontact, diffusion, uptake, or other processes well known to thoseskilled in the art. The direct activities and effects of the MAPCs maybe limited spatially, such as to an area of local deposition or to abodily compartment accessed by injection.

MAPCs also can “home” in response to “homing” signals, such as thosereleased at sites of injury or disease. Since homing often is mediatedby signals whose natural function is to recruit cells to the sites whererepairs are needed, the homing behavior can be a powerful tool forconcentrating MAPCs to therapeutic targets. This effect can bestimulated by specific factors, as discussed below.

MAPCs may also modulate immune processes by their response to factors.This may occur additionally or alternatively to direct modulation. Suchfactors may include homing factors, mitogens, and other stimulatoryfactors. They may also include differentiation factors, and factors thattrigger particular cellular processes. Among the latter are factors thatcause the secretion by cells of other specific factors, such as thosethat are involved in recruiting cells, such as stem cells (includingMAPCs), to a site of injury or disease.

MAPCs may, in addition to the foregoing or alternatively thereto,secrete factors that act on endogenous cells, such as stem cells orprogenitor cells. The factors may act on other cells to engender,enhance, decrease, or suppress their activities. MAPCs may secretefactors that act on stem, progenitor, or differentiated cells causingthose cells to divide and/or differentiate. MAPCs that home to a sitewhere repair is needed may secrete trophic factors that attract othercells to the site. In this way, MAPCs may attract stem, progenitor, ordifferentiated cells to a site where they are needed. MAPCs also maysecrete factors that cause such cells to divide or differentiate.

Secretion of such factors, including trophic factors, can contribute tothe efficacy of MAPCs in, for instance, limiting inflammatory damage,limiting vascular permeability, improving cell survival, and engenderingand/or augmenting homing of repair cells to sites of damage. Suchfactors also may affect T-cell proliferation directly. Such factors alsomay affect dendritic cells, by decreasing their phagocytic and antigenpresenting activities, which also may affect T-cell activity

By these and other mechanisms, MAPCs can provide beneficialimmunomodulatory effects, including, but not limited to, suppression ofundesirable and/or deleterious immune reactions, responses, functions,diseases, and the like. MAPCs in various embodiments of the inventionprovide beneficial immunomodulatory properties and effects that areuseful by themselves or in adjunctive therapy for precluding,preventing, lessening, decreasing, ameliorating, mitigating, treating,eliminating and/or curing deleterious immune processes and/orconditions. Such processes and conditions include, for instance,autoimmune diseases, anemias, neoplasms, HVG, GVHD, and certaininflammatory disorders, as described in greeter detail elsewhere herein.MAPCs are useful in these other regards particularly in mammals. Invarious embodiments of the invention in this regard, MAPCs are usedtherapeutically in human patients, often adjunctively to othertherapies.

MAPC Administration

MAPC Preparations

MAPCs can be prepared from a variety of tissues, such as bone marrowcells, as discussed in greater detail elsewhere herein. Initially thetissue isolates from which the MAPCs are isolated comprise a mixedpopulations of cells. MAPCs constitute a very small percentage in theseinitial populations. They must be purified away from the other cellsbefore they can be expanded in culture sufficiently to obtain enoughcells for therapeutic applications.

In many embodiments the MAPC preparations are clonally derived. Inprinciple, the MAPCs in these preparations are genetically identical toone another and, if properly prepared and maintained, are free of othercells.

In some embodiments MAPC preparations that are less pure than these maybe used. While rare, less pure populations may arise when the initialcloning step requires more than one cell. If these are not all MAPCs,expansion will produce a mixed population in which MAPCs are only one ofat least two types of cells. More often mixed populations arise whenMAPCs are administered in admixture with one or more other types ofcells.

In many embodiments the purity of MAPCs for administration to a subjectis about 100%. In other embodiments it is 95% to 100%. In someembodiments it is 85% to 95%. Particularly in the case of admixtureswith other cells, the percentage of MAPCs can be 25%-30%, 30%-35%,35%-40%, 40%-45%, 45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%.

The number of MAPCs in a given volume can be determined by well knownand routine procedures and instrumentation. The percentage of MAPCs in agiven volume of a mixture of cells can be determined by much the sameprocedures. Cells can be readily counted manually or by using anautomatic cell counter. Specific cells can be determined in a givenvolume using specific staining and visual examination and by automatedmethods using specific binding reagent, typically antibodies,fluorescent tags, and a fluorescence activated cell sorter.

MAPC immunomodulation may involve undifferentiated MAPCs. It may involveMAPCs that are committed to a differentiation pathway. Suchimmunomodulation also may involve MAPCs that have differentiated into aless potent stem cell with limited differentiation potential. It alsomay involve MAPCs that have differentiated into a terminallydifferentiated cell type. The best type or mixture of MAPCs will bedetermined by the particular circumstances of their use, and it will bea matter of routine design for those skilled in the art to determine aneffective type or combination of MAPCs.

Formulations

The choice of formulation for administering MAPCs for a givenapplication will depend on a variety of factors. Prominent among thesewill be the species of subject, the nature of the disorder, dysfunction,or disease being treated and its state and distribution in the subject,the nature of other therapies and agents that are being administered,the optimum route for administration of the MAPCs, survivability ofMAPCs via the route, the dosing regimen, and other factors that will beapparent to those skilled in the art. In particular, for instance, thechoice of suitable carriers and other additives will depend on the exactroute of administration and the nature of the particular dosage form,for example, liquid dosage form (e.g., whether the composition is to beformulated into a solution, a suspension, gel or another liquid form,such as a time release form or liquid-filled form).

For example, cell survival can be an important determinant of theefficacy of cell-based therapies. This is true for both primary andadjunctive therapies. Another concern arises when target sites areinhospitable to cell seeding and cell growth. This may impede access tothe site and/or engraftment there of therapeutic MAPCs. Variousembodiments of the invention comprise measures to increase cell survivaland/or to overcome problems posed by barriers to seeding and/or growth.

Examples of compositions comprising MAPCs include liquid preparations,including suspensions and preparations for intramuscular or intravenousadministration (e.g., injectable administration), such as sterilesuspensions or emulsions. Such compositions may comprise an admixture ofMAPCs with a suitable carrier, diluent, or excipient such as sterilewater, physiological saline, glucose, dextrose, or the like. Thecompositions can also be lyophilized. The compositions can containauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts, such as“REMINGTON′S PHARMACEUTICAL SCIENCE,” 17th edition, 1985, incorporatedherein by reference, may be consulted to prepare suitable preparations,without undue experimentation.

Compositions of the invention often are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,or viscous compositions, which may be buffered to a selected pH. Liquidpreparations are normally easier to prepare than gels, other viscouscompositions, and solid compositions. Additionally, liquid compositionsare somewhat more convenient to administer, especially by injection.Viscous compositions, on the other hand, can be formulated within theappropriate viscosity range to provide longer contact periods withspecific tissues.

Various additives often will be included to enhance the stability,sterility, and isotonicity of the compositions, such as antimicrobialpreservatives, antioxidants, chelating agents, and buffers, amongothers. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents that delayabsorption, for example, aluminum monostearate, and gelatin. Accordingto the present invention, however, any vehicle, diluent, or additiveused would have to be compatible with the cells.

MAPC solutions, suspensions, and gels normally contain a major amount ofwater (preferably purified, sterilized water) in addition to the cells.Minor amounts of other ingredients such as pH adjusters (e.g., a basesuch as NaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents and jelling agents (e.g., methylcellulose)may also be present.

Typically, the compositions will be isotonic, i.e., they will have thesame osmotic pressure as blood and lacrimal fluid when properly preparedfor administration.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycol,or other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Theimportant point is to use an amount, which will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative or cell stabilizer can beemployed to increase the life of MAPC compositions. If suchpreservatives are included, it is well within the purview of the skilledartisan to select compositions that will not affect the viability orefficacy of the MAPCs.

Those skilled in the art will recognize that the components of thecompositions should be chemically inert. This will present no problem tothose skilled in chemical and pharmaceutical principles. Problems can bereadily avoided by reference to standard texts or by simple experiments(not involving undue experimentation) using information provided by thedisclosure, the documents cited herein, and generally available in theart.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired.

In some embodiments, MAPCs are formulated in a unit dosage injectableform, such as a solution, suspension, or emulsion. Pharmaceuticalformulations suitable for injection of MAPCs typically are sterileaqueous solutions and dispersions. Carriers for injectable formulationscan be a solvent or dispersing medium containing, for example, water,saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), andsuitable mixtures thereof.

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions to beadministered in methods of the invention. Typically, any additives (inaddition to the cells) are present in an amount of 0.001 to 50 wt % insolution, such as in phosphate buffered saline. The active ingredient ispresent in the order of micrograms to milligrams, such as about 0.0001to about 5 wt %, preferably about 0.0001 to about 1 wt %, mostpreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt %, and most preferably about0.05 to about 5 wt %.

For any composition to be administered to an animal or human, and forany particular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD50 in a suitable animal model, e.g., rodent such as mouse or rat; and,the dosage of the composition(s), concentration of components therein,and timing of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure, and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

In some embodiments MAPCs are encapsulated for administration,particularly where encapsulation enhances the effectiveness of thetherapy, or provides advantages in handling and/or shelf life.Encapsulation in some embodiments where it increases the efficacy ofMAPC mediated immunosuppression may, as a result, also reduce the needfor immunosuppressive drug therapy.

Also, encapsulation in some embodiments provides a barrier to asubject's immune system that may further reduce a subject's immuneresponse to the MAPCs (which generally are not immunogenic or are onlyweakly immunogenic in allogeneic transplants), thereby reducing anygraft rejection or inflammation that might occur upon administration ofthe cells.

In a variety of embodiments where MAPCs are administered in admixturewith cells of another type, which are more typically immunogenic in anallogeneic or xenogeneic setting, encapsulation may reduce or eliminateadverse host immune responses to the non-MAPC cells and/or GVHD thatmight occur in an immunocompromised host if the admixed cells areimmunocompetent and recognize the host as non-self.

MAPCs may be encapsulated by membranes, as well as capsules, prior toimplantation. It is contemplated that any of the many methods of cellencapsulation available may be employed. In some embodiments, cells areindividually encapsulated. In some embodiments, many cells areencapsulated within the same membrane. In embodiments in which the cellsare to be removed following implantation, a relatively large sizestructure encapsulating many cells, such as within a single membrane,may provide a convenient means for retrieval.

A wide variety of materials may be used in various embodiments formicroencapsulation of MAPCs. Such materials include, for example,polymer capsules, alginate-poly-L-lysine-alginate microcapsules, bariumpoly-L-lysine alginate capsules, barium alginate capsules,polyacrylonitrile/polyvinylchloride (PAN/PVC) hollow fibers, andpolyethersulfone (PES) hollow fibers.

Techniques for microencapsulation of cells that may be used foradministration of MAPCs are known to those of skill in the art and aredescribed, for example, in Chang, P., et al., 1999; Matthew, H. W., etal., 1991; Yanagi, K., et al., 1989; Cai Z. H., et al., 1988; Chang, T.M., 1992 and in U.S. Pat. No. 5,639,275 (which, for example, describes abiocompatible capsule for long-term maintenance of cells that stablyexpress biologically active molecules. Additional methods ofencapsulation are in European Patent Publication No. 301,777 and U.S.Pat. Nos. 4,353,888; 4,744,933; 4,749,620; 4,814,274; 5,084,350;5,089,272; 5,578,442; 5,639,275; and 5,676,943. All of the foregoing areincorporated herein by reference in parts pertinent to encapsulation ofMAPCs.

Certain embodiments incorporate MAPCs into a polymer, such as abiopolymer or synthetic polymer. Examples of biopolymers include, butare not limited to, fibronectin, fibin, fibrinogen, thrombin, collagen,and proteoglycans. Other factors, such as the cytokines discussed above,can also be incorporated into the polymer. In other embodiments of theinvention, MAPCs may be incorporated in the interstices of athree-dimensional gel. A large polymer or gel, typically, will besurgically implanted. A polymer or gel that can be formulated in smallenough particles or fibers can be administered by other common, moreconvenient, non-surgical routes.

Pharmaceutical compositions of the invention may be prepared in manyforms that include tablets, hard or soft gelatin capsules, aqueoussolutions, suspensions, and liposomes and other slow-releaseformulations, such as shaped polymeric gels. Oral liquid pharmaceuticalcompositions may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups, or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid pharmaceutical compositions may containconventional additives such as suspending agents, emulsifying agents,non-aqueous vehicles (which may include edible oils), or preservatives.An oral dosage form may be formulated such that cells are released intothe intestine after passing through the stomach. Such formulations aredescribed in U.S. Pat. No. 6,306,434 and in the references containedtherein.

Pharmaceutical compositions suitable for rectal administration can beprepared as unit dose suppositories. Suitable carriers include salinesolution and other materials commonly used in the art.

For administration by inhalation, cells can be conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, a meansmay take the form of a dry powder composition, for example, a powder mixof a modulator and a suitable powder base such as lactose or starch. Thepowder composition may be presented in unit dosage form in, for example,capsules or cartridges or, e.g., gelatin or blister packs from which thepowder may be administered with the aid of an inhalator or insufflator.For intra-nasal administration, cells may be administered via a liquidspray, such as via a plastic bottle atomizer.

Other Active Ingredients

MAPCs may be administered with other pharmaceutically active agents. Insome embodiments one or more of such agents are formulated together withMAPCs for administration. In some embodiments the MAPCs and the one ormore agents are in separate formulations. In some embodiments thecompositions comprising the MAPCs and/or the one or more agents areformulated with regard to adjunctive use with one another.

MAPCs may be administered in a formulation comprising aimmunosuppressive agents, such as any combination of any number of acorticosteroid, cyclosporin A, a cyclosporin-like immunosuppressiveagent, cyclophosphamide, antithymocyte globulin, azathioprine,rapamycin, FK-506, and a macrolide-like immunosuppressive agent otherthan FK-506 and rapamycin. In certain embodiments, such agents include acorticosteroid, cyclosporin A, azathioprine, cyclophosphamide,rapamycin, and/or FK-506. Immunosuppressive agents in accordance withthe foregoing may be the only such additional agents or may be combinedwith other agents, such as other agents noted herein. Otherimmunosuppressive agents include Tacrolimus, Mycophenolate mofetil, andSirolimus.

Such agents also include antibiotic agents, antifungal agents, andantiviral agents, to name just a few other pharmacologically activesubstances and compositions that may be used in accordance withembodiments of the invention.

Typical antibiotics or anti-mycotic compounds include, but are notlimited to, penicillin, streptomycin, amphotericin, ampicillin,gentamicin, kanamycin, mycophenolic acid, nalidixic acid, neomycin,nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin,tetracycline, tylosin, zeocin, and cephalosporins, aminoglycosides, andechinocandins.

Further additives of this type relate to the fact that MAPCs, like otherstems cells, following administration to a subject may “home” to anenvironment favorable to their growth and function. Such “homing” oftenconcentrates the cells at sites where they are needed, such as sites ofimmune disorder, dysfunction, or disease. A number of substances areknown to stimulate homing. They include growth factors and trophicsignaling agents, such as cytokines. They may be used to promote homingof MAPCs to therapeutically targeted sites. They may be administered toa subject prior to treatment with MAPCs, together with MAPCs, or afterMAPCs are administered.

Certain cytokines, for instance, alter or affect the migration of MAPCsor their differentiated counterparts to sites in need of therapy, suchas immunocompromised sites. Cytokines that may be used in this regardinclude, but are not limited to, stromal cell derived factor-1 (SDF-1),stem cell factor (SCF), angiopoietin-1, placenta-derived growth factor(PIGF), granulocyte-colony stimulating factor (G-CSF), cytokines thatstimulate expression of endothelial adhesion molecules such as ICAMs andVCAMs, and cytokines that engender or facilitate homing.

They may be administered to a subject as a pre-treatment, along withMAPCs, or after MAPCs have been administered, to promote homing todesired sites and to achieve improved therapeutic effect, either byimproved homing or by other mechanisms. Such factors may be combinedwith MAPCs in a formulation suitable for them to be administeredtogether. Alternatively, such factors may be formulated and administeredseparately.

Order of administration, formulations, doses, frequency of dosing, androutes of administration of factors (such as the cytokines discussedabove) and MAPCs generally will vary with the disorder or disease beingtreated, its severity, the subject, other therapies that are beingadministered, the stage of the disorder or disease, and prognosticfactors, among others. General regimens that have been established forother treatments provide a framework for determining appropriate dosingin MAPC-mediated direct or adjunctive therapy. These, together with theadditional information provided herein, will enable the skilled artisanto determine appropriate administration procedures in accordance withembodiments of the invention, without undue experimentation.

Routes

MAPCs can be administered to a subject by any of a variety of routesknown to those skilled in the art that may be used to administer cellsto a subject.

Among methods that may be used in this regard in embodiments of theinvention are methods for administering MAPCs by a parenteral route.Parenteral routes of administration useful in various embodiments of theinvention include, among others, administration by intravenous,intraarterial, intracardiac, intraspinal, intrathecal, intraosseous,intraarticular, intrasynovial, intracutaneous, intradermal,subcutaneous, and/or intramuscular injection. In some embodimentsintravenous, intraarterial, intracutaneous, intradermal, subcutaneousand/or intramuscular injection are used. In some embodimentsintravenous, intraarterial, intracutaneous, subcutaneous, and/orintramuscular injection are used.

In various embodiments of the invention MAPCs are administered bysystemic injection. Systemic injection, such as intravenous injection,offers one of the simplest and least invasive routes for administeringMAPCs. In some cases, these routes may require high MAPC doses foroptimal effectiveness and/or homing by the MAPCs to the target sites. Ina variety of embodiments MAPCs may be administered by targeted and/orlocalized injections to ensure optimum effect at the target sites.

MAPCs may be administered to the subject through a hypodermic needle bya syringe in some embodiments of the invention. In various embodiments,MAPCs are administered to the subject through a catheter. In a varietyof embodiments, MAPCs are administered by surgical implantation. Furtherin this regard, in various embodiments of the invention, MAPCs areadministered to the subject by implantation using an arthroscopicprocedure. In some embodiments MAPCs are administered to the subject inor on a solid support, such as a polymer or gel. In various embodiments,MAPCs are administered to the subject in an encapsulated form.

In additional embodiments of the invention, MAPCs are suitablyformulated for oral, rectal, epicutaneous, ocular, nasal, and/orpulmonary delivery and are administered accordingly.

Dosing

Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the formulation that will be administered (e.g.,solid vs. liquid). Doses for humans or other mammals can be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

The dose of MAPCs appropriate to be used in accordance with variousembodiments of the invention will depend on numerous factors. It mayvary considerably for different circumstances. The parameters that willdetermine optimal doses of MAPCs to be administered for primary andadjunctive therapy generally will include some or all of the following:the disease being treated and its stage; the species of the subject,their health, gender, age, weight, and metabolic rate; the subject'simmunocompetence; other therapies being administered; and expectedpotential complications from the subject's history or genotype. Theparameters may also include: whether the MAPCs are syngeneic,autologous, allogeneic, or xenogeneic; their potency (specificactivity); the site and/or distribution that must be targeted for theMAPCs to be effective; and such characteristics of the site such asaccessibility to MAPCs and/or engraftment of MAPCs. Additionalparameters include co-administration with MAPCs of other factors (suchas growth factors and cytokines). The optimal dose in a given situationalso will take into consideration the way in which the cells areformulated, the way they are administered, and the degree to which thecells will be localized at the target sites following administration.Finally, the determination of optimal dosing necessarily will provide aneffective dose that is neither below the threshold of maximal beneficialeffect nor above the threshold where the deleterious effects associatedwith the dose of MAPCs outweighs the advantages of the increased dose.

The optimal dose of MAPCs for some embodiments will be in the range ofdoses used for autologous, mononuclear bone marrow transplantation. Forfairly pure preparations of MAPCs, optimal doses in various embodimentswill range from 10⁴ to 10⁸ MAPC cells/kg of recipient mass peradministration. In some embodiments the optimal dose per administrationwill be between 10⁵ to 10⁷ MAPC cells/kg. In many embodiments theoptimal dose per administration will be 5×10⁵ to 5×10⁶ MAPC cells/kg. Byway of reference, higher doses in the foregoing are analogous to thedoses of nucleated cells used in autologous mononuclear bone marrowtransplantation. Some of the lower doses are analogous to the number ofCD34⁺ cells/kg used in autologous mononuclear bone marrowtransplantation.

It is to be appreciated that a single dose may be delivered all at once,fractionally, or continuously over a period of time. The entire dosealso may be delivered to a single location or spread fractionally overseveral locations.

In various embodiments, MAPCs may be administered in an initial dose,and thereafter maintained by further administration of MAPCs. MAPCs maybe administered by one method initially, and thereafter administered bythe same method or one or more different methods. The subject's MAPClevels can be maintained by the ongoing administration of the cells.Various embodiments administer the MAPCs either initially or to maintaintheir level in the subject or both by intravenous injection. In avariety of embodiments, other forms of administration, are used,dependent upon the patient's condition and other factors, discussedelsewhere herein.

It is noted that human subjects are treated generally longer thanexperimental animals; but, treatment generally has a length proportionalto the length of the disease process and the effectiveness of thetreatment. Those skilled in the art will take this into account in usingthe results of other procedures carried out in humans and/or in animals,such as rats, mice, non-human primates, and the like, to determineappropriate doses for humans. Such determinations, based on theseconsiderations and taking into account guidance provided by the presentdisclosure and the prior art will enable the skilled artisan to do sowithout undue experimentation.

Suitable regimens for initial administration and further doses or forsequential administrations may all be the same or may be variable.Appropriate regiments can be ascertained by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

The dose, frequency, and duration of treatment will depend on manyfactors, including the nature of the disease, the subject, and othertherapies that may be administered. Accordingly, a wide variety ofregimens may be used to administer MAPCs.

In some embodiments MAPCs are administered to a subject in one dose. Inothers MAPCs are administered to a subject in a series of two or moredoses in succession. In some other embodiments wherein MAPCs areadministered in a single dose, in two doses, and/or more than two doses,the doses may be the same or different, and they are administered withequal or with unequal intervals between them.

MAPCs may be administered in many frequencies over a wide range oftimes. In some embodiments, MAPCs are administered over a period of lessthan one day. In other embodiment they are administered over two, three,four, five, or six days. In some embodiments MAPCs are administered oneor more times per week, over a period of weeks. In other embodimentsthey are administered over a period of weeks for one to several months.In various embodiments they may be administered over a period of months.In others they may be administered over a period of one or more years.Generally lengths of treatment will be proportional to the length of thedisease process, the effectiveness of the therapies being applied, andthe condition and response of the subject being treated.

Therapeutic Uses of Immunomodulating MAPCs

The immunomodulatory properties of MAPCs may be used in treating a widevariety of disorders, dysfunctions and diseases, such as those that,intrinsically, as a secondary effect or as a side effect of treatment,present with deleterious immune system processes and effects. Severalillustrations are discussed below.

Many embodiments in this regard involve administering MAPCs to a subjecthaving a weakened (or compromised) immune system, either as the soletherapy or as adjunctive therapy with another treatment. In a variety ofembodiments in this regard MAPCs are administered to a subjectadjunctively to radiation therapy or chemotherapy or a combination ofradiation and chemotherapies that either have been, are being, or willbe administered to the subject. In many such embodiments, the radiationtherapy, chemotherapy, or a combination of radiation and chemotherapiesare part of a transplant therapy. And in a variety of embodiments MAPCsare administered to treat a deleterious immune response, such as HVG orGVHD.

In a variety of embodiments in this regard, the subject is the recipientof a non-syngeneic, typically allogeneic, blood cell or bone marrow celltransplant, the immune system of the subject has been weakened orablated by radiation therapy, chemotherapy, or a combination ofradiation and chemotherapy, immunosuppressive drugs are beingadministered to the subject, the subject is at risk to develop or hasdeveloped graft versus host disease, and MAPCs are administered to thesubject adjunctively to any one or more of the transplant, the radiationtherapy and/or the chemotherapy, and the immunosuppressive drugs totreat, such as ameliorate, arrest, or eliminate, graft versus hostdisease in the subject.

Neoplasms

The term “neoplasm” generally denotes disorders involving the clonalproliferation of cells. Neoplasms may be benign, which is to say, notprogressive and non-recurrent, and, if so, generally are notlife-threatening. Neoplasms also may be malignant, which is to say, thatthey progressively get worse, spread, and, as a rule, are lifethreatening and often fatal.

In various embodiments, MAPCs are administered to a subject sufferingfrom a neoplasm, adjunctive to a treatment thereof. For example, in someembodiments of the invention in this regard, the subject is at risk foror is suffering from a neoplasm of blood or bone marrow cells and hasundergone or will undergo a blood or bone marrow transplant. Using themethods described herein for MAPC isolation, characterization, andexpansion, together with the disclosures herein on immune-suppressingproperties of MAPCs, MAPCs are administered to treat, such as toprevent, suppress, or diminish, the deleterious immune reactions, suchas HVG and GVHD, that may complicate the transplantation therapy.

In a variety of embodiments involving transplant therapies, MAPCs can beused alone for an immunosuppressive purpose, or together with otheragents. MAPCs can be administered before, during, or after one or moretransplants. If administered during transplant, MAPCs can beadministered separately or together with transplant material. Ifseparately administered, the MAPCs can be administered sequentially orsimultaneously with the other transplant materials. Furthermore, MAPCsmay be administered well in advance of the transplant and/or well after,alternatively to or in addition to administration at or about the sametime as administration of the transplant.

Other agents that can be used in conjunction with MAPCs, intransplantation therapies in particular, include immunomodulatoryagents, such as those described elsewhere herein, particularlyimmunosuppressive agents, more particularly those described elsewhereherein, especially in this regard, one or more of a corticosteroid,cyclosporin A, a cyclosporin-like immunosuppressive compound,azathioprine, cyclophosphamide, methotrexate, and an immunosuppressivemonoclonal antibody agent.

Among neoplastic disorders of bone marrow that are treated with MAPCs inembodiments of the invention in this regard are myeloproliferativedisorders (“MPDs”); myelodysplastic syndromes (or states) (“MDSs”),leukemias, and lymphoproliferative disorders including multiple myelomaand lymphomas.

MPDs are distinguished by aberrant and autonomous proliferation of cellsin blood marrow. The disorder may involve only one type of cell orseveral. Typically, MPDs involve three cell lineages and areerythrocytic, granulocytic, and thrombocytic. Involvement of the threelineages varies from one MPD to another and between occurrences of theindividual types. Typically, they are differently affected and one celllineage is affected predominately in a given neoplasm. MPDs are notclearly malignant; but, they are classified as neoplasms and arecharacterized by aberrant, self-replication of hematopoietic precursorcells in blood marrow. MPDs have the potential, nonetheless, to developinto acute leukemias.

MDSs like MPDs are clonal disorders, and they are characterized byaberrant, self-replication of hematopoietic precursor cells in bloodmarrow. Like MPDs, they can develop into acute leukemias. Most, but notall, MDSs manifest peripheral blood cytopenias (chronic myelomonocyticleukemia is the exception), whereas MPDs do not.

The laboratory and clinical manifestations of these disorders may varywith their course and between individual occurrences. Manifestations canoverlap, and it can be difficult to make a certain diagnosis thatdistinguishes one disease from all the others. Diagnosis of neoplasms ofbone marrow hematopoietic cells thus requires special caution, so as tonot misdiagnose as a benign disorder one that is, in reality, deadlymalignant.

The following diseases are among the myeloproliferative disorders (MPDs)that may be treated with MAPCs, typically or adjunctively, in variousembodiments of the invention: chronic myelocytic leukemia(“CML”)/chronic granulocytic leukemia (“CGL”), agnogenic myelofibrosis,essential thrombocythemia, and polycythemia vera

The following diseases are among the myelodysplastic syndromes (MDSs)that may be treated with MAPCs, typically or adjunctively, in variousembodiments of the invention: refractory anemia, refractory anemia withringed sideroblasts, refractory anemia with excess blasts, refractoryanemia with excess blasts in transformation, and chronic myelomonocyticleukemia.

The following diseases are among the lymphoproliferative disorders,including multiple myelomas and lymphomas that may be treated withMAPCs, typically adjunctively, in various embodiments of the invention:pre-B acute lymphoblastic leukemia, chronic lymphocytic leukemia(“CLL”), B-cell lymphoma, hairy cell leukemia, myeloma, multiplemyeloma, T-acute lymphoblastic leukemia, peripheral T-cell lymphoma,other lymphoid leukemias, and other lymphomas.

Also among neoplasms that may be treated with MAPCs, typicallyadjunctively, in a variety of embodiments of the invention are thefollowing: a benign neoplasm of bone marrow cells, a myeloproliferativedisorder, a myelodysplastic syndrome, or an acute leukemia; chronicmyelocytic leukemia (“CML”) (also called chronic granulocytic leukemia(“CGL”)), agnogenic myelofibrosis, essential thrombocythemia,polycythemia vera, other myeloproliferative disorders, acute multiplemyeloma, myeloblastic leukemia, acute promyelocytic leukemia, pre-Bacute lymphoblastic leukemia, chronic lymphocytic leukemia (“CLL”),B-cell lymphoma, hairy cell leukemia, myeloma, T-acute lymphoblasticleukemia, peripheral T-cell lymphoma, other lymphoid leukemias, otherlymphomas, or other acute leukemia.

MAPCs may be administered adjunctively to a treatment for any of theforegoing diseases.

Treatments Involving Immunoablation or Compromise

Acute leukemias often are difficult to treat by methods that have beeneffective for other malignancies. This is partly due to the mobility ofcells from bone marrow, including those of a neoplasm. Partly it may bedue to the diffuse distribution of bone marrow throughout the skeleton.And partly it is due, no doubt, to the nature of the cells themselvesand their transformation.

At present, a standard treatment for hematologic malignancies involvesablating all hematopoietic cells in the patient. There is no way to dothis without also ablating the patient's healthy hematopoietic cells.Typically, the patient is treated using chemo-radiotherapy insufficiently high doses to kill virtually all bone marrow cells, bothnormal and neoplastic. The treatment's side effects are severe, and itseffects on patients are unpleasant, painful, and physically andemotionally debilitating. The treatment not only ablates the diseasedtissue and cells, it also eviscerates the patient's hematopoietic systemand immune system. The treatment leaves them compromised, dependent ontransfusions, and so highly susceptible to infection that even anotherwise minor exposure to an infectious agent can be fatal.

Normal hematopoietic capacity is restored thereafter by eitherautologous or allogeneic peripheral blood or bone marrow transplants.Unfortunately, the patient's immune system not only is severelycompromised by the ablative treatment, but, also in the case ofallogeneic transplantation, by intentional immune suppression to preventrejection of the transplant and ensure engraftment and proliferation ofthe new hematopoietic stem cells that will repopulate the patient'smarrow and regenerate the patient's hematopoietic and immune systems.

Many complications have been encountered in carrying out suchtransplants to regenerate the hematopoietic and immune systems in animmunocompromised host. One is rejection by residual immune competentcells and processes in the host, referred to herein as HVG response.Another is triggered by immunocompetent cells in the graft, referred toherein as GVHD.

These complications might be avoided by using syngeneic or autologousdonor material. However, syngeneic donors generally are rare andautologous transplants have a high risk of disease recurrence. Hence,transplants generally use allogeneic cells and tissues obtained from anHLA compatible donor. Unfortunately this procedure results in GVHD,ranging from mild to severe in the preponderance of those receiving thisform of therapy. If not at least ameliorated, these immune reactionswill result in failure of the transplant therapy, and may themselves befatal to the patient.

A variety of agents have been developed to suppress immune responsesthat ameliorate graft complications, such as HVG and GVHD, as discussedherein above. Some are sufficiently effective to reduce adverse immunereactions to a manageable level in some transplant therapies, such asbone marrow and peripheral blood transplantation. These agents haveimproved the prognosis for transplant patients, to some extent; but,none of them is fully effective, and all of them have rather substantialshortcomings.

It has been found (as described in greater detail elsewhere herein) thatMAPCs do not provoke an immune response in allogeneic hosts.Transplantation of MAPCs to an allogeneic host does not, thus, engenderallogeneic graft rejection (i.e., HVG).

Furthermore, it has also been found that allogeneic MAPCs can beadministered to a host at high concentration without deleterious effectson respiration.

In addition, it has been found, (as described in greater detailelsewhere herein) that MAPCs can modulate immune responses. Inparticular in this regard, it has been found that MAPCs can suppressimmune responses, including but not limited to immune responses involvedin, for example, HVG response and GVHD, to name just two. In an evenmore detailed particular in this regard, it has been found that MAPCscan suppress proliferation of T-cells, even in the presence of potentT-cell stimulators, such as Concanavalin A and allogeneic or xenogeneicstimulator cells.

Moreover, it has been found that even relatively small amounts of MAPCscan suppress these responses. Indeed, only 3% MAPCs in mixed lymphocytereactions is sufficient to reduce T-cell response by 50% in vitro.

Accordingly, embodiments of the invention provide compositions andmethods and the like for treating, such as for ameliorating, and/orcuring or eliminating, neoplasms, such as neoplasms of hematopoieticcells, particularly those of bone marrow.

Among these are those that are myeloproliferative disorders (MPDs), suchas chronic myelocytic leukemia (also called “chronic granulocyticleukemia” and “CGL”), agnogenic myelofibrosis, essentialthrombocythemia, polycythemia vera; myelodysplastic syndromes (MDSs),such as refractory anemia, refractory anemia with ringed sideroblasts,refractory anemia with excess blasts, refractory anemia with excessblasts in transformation, chronic myelomonocytic leukemia; and clearlymalignant neoplasms—the acute leukemias—such as acute myeloblasticleukemia, chronic myelogenous leukemia (CML), acute promyelocyticleukemia, B-acute lymphoblastic leukemia, CLL, B-cell lymphoma, hairycell leukemia, myeloma, T-acute lymphoblastic leukemia, peripheralT-cell lymphoma, and other lymphoid leukemias and lymphomas.

MAPCs may be administered adjunctively to a treatment for any of theforegoing diseases.

Anemias and Other Disorders of the Blood

In various embodiments of the invention, MAPCs may be used to treat ananemia or other blood disorder, often adjunctively. Among variousembodiments in this regard are embodiments in which MAPCs are used totreat the following anemias and/or blood disorders, either solely or,typically, adjunctively: hemoglobinopathies, thalassemia, bone marrowfailure syndrome, sickle cell anemia, aplastic anemia, or an immunehemolytic anemia. Also, disorders include refractory anemia, refractoryanemia with ringed sideroblasts, refractory anemia with excess blasts,refractory anemia with excess blasts in transformation, chronicmyelomonocytic leukemia, or other myelodysplastic syndrome, and in someembodiments, Fanconi's anemia.

MAPCs may be administered adjunctively to a treatment for any of theforegoing diseases.

Immune Diseases

Embodiments of the invention relate to using MAPC immunomodulation totreat an immune dysfunction, disorder, or disease, either solely, or asan adjunctive therapy. Embodiments in this regard relate to congenitalimmune deficiencies and autoimmune dysfunctions, disorders, anddiseases. Various embodiments relate, in this regard, to using MAPCs totreat, solely or adjunctively, Crohn's disease, Guillain-Barré syndrome,lupus erythematosus (also called “SLE” and systemic lupuserythematosus), multiple sclerosis, myasthenia gravis, optic neuritis,psoriasis, rheumatoid arthritis, Graves' disease, Hashimoto's disease,Ord's thyroiditis, diabetes mellitus (type 1), Reiter's syndrome,autoimmune hepatitis, primary biliary cirrhosis, antiphospholipidantibody syndrome (“APS”), opsoclonus-myoclonus syndrome (“OMS”),temporal arteritis, acute disseminated encephalomyelitis (“ADEM” and“ADE”), Goodpasture's, syndrome, Wegener's granulomatosis, celiacdisease, pemphigus, polyarthritis, and warm autoimmune hemolytic anemia.

Particular embodiments among these relate to Crohn's disease, lupuserythematosus (also called “SLE” and systemic lupus erythematosus),multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis,Graves' disease, Hashimoto's disease, diabetes mellitus (type 1),Reiter's syndrome, primary biliary cirrhosis, celiac disease,polyarthritis, and warm autoimmune hemolytic anemia.

In addition, MAPCs are used in a variety of embodiments in this regard,solely and, typically, adjunctively, to treat a variety of diseasesthought to have an autoimmune component, including but not limited toembodiments that may be used to treat endometriosis, interstitialcystitis, neuromyotonia, scleroderma, progressive systemic scleroderma,vitiligo, vulvodynia, Chagas' disease, sarcoidosis, chronic fatiguesyndrome, and dysautonomia.

Inherited immune system disorders include Severe CombinedImmunodeficiency (SCID) including but not limited to SCID with AdenosineDeaminase Deficiency (ADA-SCID), SCID which is X-linked, SCID withabsence of T & B Cells, SCID with absence of T Cells, Normal B Cells,Omenn Syndrome, Neutropenias including but not limited to KostmannSyndrome, Myelokathexis; Ataxia-Telangiectasia, Bare LymphocyteSyndrome, Common Variable Immunodeficiency, DiGeorge Syndrome, LeukocyteAdhesion Deficiency; and phagocyte Disorders (phagocytes are immunesystem cells that can engulf and kill foreign organisms) including butnot limited to Chediak-Higashi Syndrome, Chronic Granulomatous Disease,Neutrophil Actin Deficiency, Reticular Dysgenesis.

MAPCs may be administered adjunctively to a treatment for any of theforegoing diseases.

Inflammatory Diseases

Additionally, in a variety of embodiments of the invention, MAPCs may beused to treat inflammatory diseases, either as a sole agent oradjunctively. In many such embodiments MAPCs may be used to treatserious inflammatory states, such as those that arise from acuteallergic reaction, or ancillary to other diseases or treatments. For themost part, at present, the use of MAPCs in this regard is limited toacute cases in which the subject is at risk of great incapacity or lossor life.

MAPCs may be administered adjunctively to a treatment for any of theforegoing diseases.

MAPCs as Described in PCT/US00/21387

Human MAPCs are described in U.S. patent application Ser. Nos.10/048,757 (PCT/US00/21387 (published as WO 01/11011)) and Ser. No.10/467,963 (PCT/US02/04652 (published as WO 02/064748)), the contents ofwhich are incorporated herein by reference for their description ofMAPCs. MAPCs have been identified in other mammals. Murine MAPCs, forexample, are also described in PCT/US00/21387 (published as WO 01/11011)and PCT/US02/04652 (published as WO 02/064748). Rat MAPCs are alsodescribed in WO 02/064748.

Isolation and Growth of MAPCs as Described in PCT/US00/21387

Methods of MAPC isolation for humans and mouse are known in the art.They are described in PCT/US00/21387 (published as WO 01/11011) and forrat in PCT/US02/04652 (published as WO 02/064748), and these methods,along with the characterization of MAPCs disclosed therein, areincorporated herein by reference.

MAPCs were initially isolated from bone marrow, but were subsequentlyestablished from other tissues, including brain and muscle (Jiang, Y. etal., 2002). Thus, MAPCs can be isolated from multiple sources, includingbone marrow, placenta, umbilical cord and cord blood, muscle, brain,liver, spinal cord, blood or skin. For example, MAPCs can be derivedfrom bone marrow aspirates, which can be obtained by standard meansavailable to those of skill in the art (see, for example, Muschler, G.F., et al., 1997; Batinic, D., et al., 1990). It is therefore nowpossible for one of skill in the art to obtain bone marrow aspirates,brain or liver biopsies, and other organs, and isolate the cells usingpositive or negative selection techniques available to those of skill inthe art, relying upon the genes that are expressed (or not expressed) inthese cells (e.g., by functional or morphological assays such as thosedisclosed in the above-referenced applications, which have beenincorporated herein by reference).

MAPCs from Human Bone Marrow as Described in PCT/US00/21387

Bone marrow mononuclear cells were derived from bone marrow aspirates,which were obtained by standard means available to those of skill in theart (see, for example, Muschler, G. F., et al., 1997; Batinic, D., etal., 1990). Multipotent adult stem cells are present within the bonemarrow (or other organs such as liver or brain), but do not express thecommon leukocyte antigen CD45 or erythroblast specific glycophorin-A(Gly-A). The mixed population of cells was subjected to a Ficoll Hypaqueseparation. The cells were then subjected to negative selection usinganti-CD45 and anti-Gly-A antibodies, depleting the population of CD45+and Gly-A+ cells, and the remaining approximately 0.1% of marrowmononuclear cells were then recovered. Cells could also be plated infibronectin-coated wells and cultured as described below for 2-4 weeksto deplete the cells of CD45+ and Gly-A+ cells.

Alternatively, positive selection could be used to isolate cells via acombination of cell-specific markers. Both positive and negativeselection techniques are available to those of skill in the art, andnumerous monoclonal and polyclonal antibodies suitable for negativeselection purposes are also available in the art (see, for example,Leukocyte Typing V, Schlossman et al., eds. (1995) Oxford UniversityPress) and are commercially available from a number of sources.

Techniques for mammalian cell separation from a mixture of cellpopulations have also been described by Schwartz, et al., in U.S. Pat.No. 5,759,793 (magnetic separation), Basch et al., 1983 (immunoaffinitychromatography), and Wysocki and Sato, 1978 (fluorescence-activated cellsorting).

Recovered CD45⁻/GlyA⁻ cells were plated onto culture dishes coated with5-115 ng/ml (about 7-10 ng/ml can be used) serum fibronectin or otherappropriate matrix coating. Cells were maintained in Dulbecco's MinimalEssential Medium (DMEM) or other appropriate cell culture medium,supplemented with 1-50 ng/ml (about 5-15 ng/ml can be used)platelet-derived growth factor-BB (PDGF-BB), 1-50 ng/ml (about 5-15ng/ml can be used) epidermal growth factor (EGF), 1-50 ng/ml (about 5-15ng/ml can be used) insulin-like growth factor (IGF), or 100-10,000 IU(about 1,000 IU can be used) LIF, with 10⁻¹⁰ to 10⁻⁸ M dexamethasone (orother appropriate steroid), 2-10 μg/ml linoleic acid, and 0.05-0.15 μMascorbic acid. Other appropriate media include, for example, MCDB, MEM,IMDM, and RPMI. Cells can either be maintained without serum, in thepresence of 1-2% fetal calf serum, or, for example, in 1-2% human ABserum or autologous serum.

When re-seeded at 2×10³ cells/cm² every 3 days, >40 cell doublings wereroutinely obtained, and some populations underwent >70 cell doublings.Cell doubling time was 36-48 h for the initial 20-30 cell doublings.Afterwards cell-doubling time was extended to as much as 60-72 h.

Telomere length of MAPCs from 5 donors (age about 2 years to about 55years) cultured at re-seeding densities of 2×10³ cells/cm² for 23-26cell doublings was between 11-13 KB. This was 3-5 KB longer thantelomere length of blood lymphocytes obtained from the same donors.Telomere length of cells from 2 donors evaluated after 23 and 25 celldoublings, respectively, and again after 35 cells doublings, wasunchanged. The karyotype of these MAPCs was normal.

Phenotype of Human MAPCs Under Conditions Described in PCT/US00/21387

Immunophenotypic analysis by FACS of human MAPCs obtained after 22-25cell doublings indicated that the cells do not express CD31, CD34, CD36,CD38, CD45, CD50, CD62E and -P, HLA-DR, Muc18, STRO-1, cKit, Tie/Tek;and express low levels of CD44, HLA-class I, and β2-microglobulin, butexpress CD10, CD13, CD49b, CD49e, CDw90, Flk1 (N>10).

Once cells underwent >40 doublings in cultures re-seeded at about2×10³/cm², the phenotype became more homogenous, and no cell expressedHLA class-I or CD44 (n=6).

When cells were grown at higher confluence, they expressed high levelsof Muc18, CD44, HLA class I and β2-microglobulin, which is similar tothe phenotype described for MSC (N=8) (Pittenger, 1999).

Immunohistochemistry showed that human MAPCs grown at about 2×10³/cm²seeding density expressed EGF-R, TGF-R1 and -2, BMP-R1A, PDGF-R1a and-B, and that a small subpopulation (between 1 and 10%) of MAPCs stainedwith anti-SSEA4 antibodies (Kannagi, R, 1983).

Using Clontech cDNA arrays the expressed gene profile of human MAPCscultured at seeding densities of about 2×10³ cells/cm² for 22 and 26cell doublings was determined:

A. MAPCs did not express CD31, CD36, CD62E, CD62P, CD44-H, cKit, Tie,receptors for IL1, IL3, IL6, IL11, G CSF, GM-CSF, Epo, Flt3-L, or CNTF,and low levels of HLA-class-I, CD44-E and Muc-18 mRNA.

B. MAPCs expressed mRNA for the cytokines BMP1, BMP5, VEGF, HGF, KGF,MCP1; the cytokine receptors Flk1, EGF-R, PDGF-R1a, gp130, LIF-R,activin-R1 and -R2, TGFR-2, BMP-R1A; the adhesion receptors CD49c,CD49d, CD29; and CD10.

C. MAPCs expressed mRNA for hTRT and TRF1; the POU domain transcriptionfactor oct-4, sox-2 (required with oct-4 to maintain undifferentiatedstate of ES/EC, Uwanogho D., 1995), sox 11 (neural development), sox 9(chondrogenesis) (Lefebvre V., 1998); homeodeomain transcriptionfactors: Hox-a4 and -a5 (cervical and thoracic skeleton specification;organogenesis of respiratory tract) (Packer A I, 2000), Hox-a9(myelopoiesis) (Lawrence H, 1997), Dlx4 (specification of forebrain andperipheral structures of head) (Akimenko M A, 1994), MSX1 (embryonicmesoderm, adult heart and muscle, chondro- and osteogenesis)(Foerst-Potts L. 1997), PDX1 (pancreas) (Offield M F, 1996).

D. Presence of oct-4, LIF-R, and hTRT mRNA was confirmed by RT-PCR.

E. In addition, RT-PCR showed that rex-1 mRNA and rox-1 mRNA wereexpressed in MAPCs.

Oct-4, rex-1 and rox-1 were expressed in MAPCs derived from human andmurine marrow and from murine liver and brain. Human MAPCs expressedLIF-R and stained positive with SSEA-4. Finally, oct-4, LIF-R, rex-1 androx-1 mRNA levels were found to increase in human MAPCs cultured beyond30 cell doublings, which resulted in phenotypically more homogenouscells. In contrast, MAPCs cultured at high density lost expression ofthese markers. This was associated with senescence before 40 celldoublings and loss of differentiation to cells other than chondroblasts,osteoblasts, and adipocytes. Thus, the presence of oct-4, combined withrex-1, rox-1, and sox-2, correlated with the presence of the mostprimitive cells in MAPCs cultures.

Culturing MAPCs as Described in PCT/US00/21387

MAPCs isolated as described herein can be cultured using methodsdisclosed herein and in PCT/US00/21387, which is incorporated byreference for these methods.

Briefly, for the culture of MAPCs, culture in low-serum or serum-freemedium was preferred to maintain the cells in the undifferentiatedstate. Serum-free medium used to culture the cells, as described herein,was supplemented as described in Table 1. Human MAPCs do not requireLIF.

TABLE 1 Insulin 10-50 μg/ml (10 μg/ml)* Transferrin 0-10 μg/ml (5.5μg/ml) Selenium 2-10 ng/ml (5 ng/ml) Bovine serum albumin (BSA) 0.1-5μg/ml (0.5 μg/ml) Linoleic acid 2-10 μg/ml (4.7 μg/ml) Dexamethasone0.005-0.15 μM (.01 μM) L-ascorbic acid 2-phosphate 0.1 mM Low-glucoseDMEM (DMEM-LG) 40-60% (60%) MCDB-201 40-60% (40%) Fetal calf serum 0-2%Platelet-derived growth 5-15 ng/ml (10 ng/ml) Epidermal growth factor5-15 ng/ml (10 ng/ml) Insulin like growth factor 5-15 ng/ml (10 ng/ml)Leukemia inhibitory factor 10-10,000 IU (1,000 IU) *Preferredconcentrations are shown in parentheses.

Addition of 10 ng/ml LIF to human MAPCs did not affect short-term cellgrowth (same cell doubling time till 25 cell doublings, level of oct-4(oct-3/4) expression). In contrast to what was seen with human cells,when fresh murine marrow mononuclear cells, depleted on day 0 of CD45⁺cells, were plated in MAPC culture, no growth was seen. When murinemarrow mononuclear cells were plated, and cultured cells 14 days laterdepleted of CD45⁺ cells, cells with the morphology and phenotype similarto that of human MAPCs appeared. This suggested that factors secreted byhemopoietic cells were needed to support initial growth of murine MAPCs.When cultured with PDGF-BB and EFG alone, cell doubling was slow (>6days) and cultures could not be maintained beyond 10 cell doublings.Addition of 10 ng/m LIF significantly enhanced cell growth.

Once established in culture, cells could be frozen and stored as frozenstocks, using DMEM with 40% FCS and 10% DMSO. Other methods forpreparing frozen stocks for cultured cells are also available to thoseof skill in the art.

Thus, MAPCs could be maintained and expanded in culture medium that isavailable to the art. Such media include, but are not limited toDulbecco's Modified Eagle's Medium® (DMEM), DMEM F12 Medium®, Eagle'sMinimum Essential Medium®, F-12K Medium®, Iscove's Modified Dulbecco'sMedium® and RPMI-1640 Medium®. Many media are also available aslow-glucose formulations, with or without sodium pyruvate.

Also contemplated is supplementation of cell culture medium withmammalian sera. Sera often contain cellular factors and components thatare necessary for viability and expansion. Examples of sera includefetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calfserum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum(HS), human serum, chicken serum, porcine serum, sheep serum, rabbitserum, serum replacements, and bovine embryonic fluid. It is understoodthat sera can be heat-inactivated at 55-65° C. if deemed necessary toinactivate components of the complement cascade.

Additional supplements can also be used advantageously to supply thecells with the necessary trace elements for optimal growth andexpansion. Such supplements include insulin, transferrin, sodiumselenium, and combinations thereof. These components can be included ina salt solution such as, but not limited to, Hanks' Balanced SaltSolution® (HBSS), Earle's Salt Solution®, antioxidant supplements,MCDB-201® supplements, phosphate buffered saline (PBS), ascorbic acidand ascorbic acid-2-phosphate, as well as additional amino acids. Manycell culture media already contain amino acids, however some requiresupplementation prior to culturing cells. Such amino acids include, butare not limited to, L-alanine, L-arginine, L-aspartic acid,L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine. It is well within the skill of one in the artto determine the proper concentrations of these supplements.

Antibiotics are also typically used in cell culture to mitigatebacterial, mycoplasmal, and fungal contamination. Typically, antibioticsor anti-mycotic compounds used are mixtures of penicillin/streptomycin,but can also include, but are not limited to amphotericin (Fungizone®),ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin,mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin,polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin,and zeocin. Antibiotic and anti-mycotic additives can be of someconcern, depending on the type of work being performed. One possiblesituation that can arise is an antibiotic-containing media whereinbacteria are still present in the culture, but the action of theantibiotic performs a bacteriostatic rather than bacteriocidalmechanism. Also, antibiotics can interfere with the metabolism of somecell types.

Hormones can also be advantageously used in cell culture and include,but are not limited to D-aldosterone, diethylstilbestrol (DES),dexamethasone, β-estradiol, hydrocortisone, insulin, prolactin,progesterone, somatostatin/human growth hormone (HGH), thyrotropin,thyroxine, and L-thyronine.

Lipids and lipid carriers can also be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Such lipids and carriers can include, but are not limited tocyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated toalbumin, linoleic acid and oleic acid conjugated to albumin,unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugatedto albumin, oleic acid unconjugated and conjugated to albumin, amongothers.

Also contemplated is the use of feeder cell layers. Feeder cells areused to support the growth of fastidious cultured cells, particularly EScells. Feeder cells are normal cells that have been inactivated byγ-irradiation. In culture, the feeder layer serves as a basal layer forother cells and supplies cellular factors without further growth ordivision of their own (Lim, J. W. and Bodnar, A., 2002). Examples offeeder layer cells are typically human diploid lung cells, mouseembryonic fibroblasts, and Swiss mouse embryonic fibroblasts, but can beany post-mitotic cell that is capable of supplying cellular componentsand factors that are advantageous in allowing optimal growth, viability,and expansion of stem cells. In many cases, feeder cell layers are notnecessary to keep the ES cells in an undifferentiated, proliferativestate, as leukemia inhibitory factor (LIF) has anti-differentiationproperties. Therefore, supplementation with LIF could be used tomaintain MAPC in some species in an undifferentiated state.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components and synthetic orbiopolymers. Stem cells often require additional factors that encouragetheir attachment to a solid support, such as type I, type II, and typeIV collagen, concanavalin A, chondroitin sulfate, fibronectin,“superfibronectin” and fibronectin-like polymers, gelatin, laminin,poly-D and poly-L-lysine, thrombospondin, and vitronectin.

The maintenance conditions of stem cells can also contain cellularfactors that allow stem cells, such as MAPCs, to remain in anundifferentiated form. It is advantageous under conditions where thecell must remain in an undifferentiated state of self-renewal for themedium to contain epidermal growth factor (EGF), platelet derived growthfactor (PDGF), leukemia inhibitory factor (LIF, in selected species),and combinations thereof. It is apparent to those skilled in the artthat supplements that allow the cell to self-renew but not differentiatemust be removed from the culture medium prior to differentiation.

Stem cell lines and other cells can benefit from co-culturing withanother cell type. Such co-culturing methods arise from the observationthat certain cells can supply yet-unidentified cellular factors thatallow the stem cell to differentiate into a specific lineage or celltype. These cellular factors can also induce expression of cell-surfacereceptors, some of which can be readily identified by monoclonalantibodies. Generally, cells for co-culturing are selected based on thetype of lineage one skilled in the art wishes to induce, and it iswithin the capabilities of the skilled artisan to select the appropriatecells for co-culture.

Methods of identifying and subsequently separating differentiated cellsfrom their undifferentiated counterparts can be carried out by methodswell known in the art. Cells that have been induced to differentiate canbe identified by selectively culturing cells under conditions wherebydifferentiated cells outnumber undifferentiated cells. Similarly,differentiated cells can be identified by morphological changes andcharacteristics that are not present on their undifferentiatedcounterparts, such as cell size, the number of cellular processes (i.e.,formation of dendrites and/or branches), and the complexity ofintracellular organelle distribution. Also contemplated are methods ofidentifying differentiated cells by their expression of specificcell-surface markers such as cellular receptors and transmembraneproteins. Monoclonal antibodies against these cell-surface markers canbe used to identify differentiated cells. Detection of these cells canbe achieved through fluorescence activated cell sorting (FACS), andenzyme-linked immunosorbent assay (ELISA). From the standpoint oftranscriptional upregulation of specific genes, differentiated cellsoften display levels of gene expression that are different fromundifferentiated cells. Reverse-transcription polymerase chain reaction(RT-PCR) can also be used to monitor changes in gene expression inresponse to differentiation. In addition, whole genome analysis usingmicroarray technology can be used to identify differentiated cells.

Accordingly, once differentiated cells are identified, they can beseparated from their undifferentiated counterparts, if necessary. Themethods of identification detailed above also provide methods ofseparation, such as FACS, preferential cell culture methods, ELISA,magnetic beads, and combinations thereof. A preferred embodiment of theinvention envisions the use of FACS to identify and separate cells basedon cell-surface antigen expression.

Additional Culture Methods

In additional experiments it has been found that the density at whichMAPCs are cultured can vary from about 100 cells/cm² or about 150cells/cm² to about 10,000 cells/cm², including about 200 cells/cm² toabout 1500 cells/cm² to about 2000 cells/cm². The density can varybetween species. Additionally, optimal density can vary depending onculture conditions and source of cells. It is within the skill of theordinary artisan to determine the optimal density for a given set ofculture conditions and cells.

Also, effective atmospheric oxygen concentrations of less than about10%, including about 3-5%, can be used at any time during the isolation,growth, and differentiation of MAPCs in culture.

Inducing MAPCs to Differentiate to Form Committed Progenitors andTissue-Specific Cell Types as Described in PCT/US00/21387

Using appropriate growth factors, chemokines, and cytokines, MAPCs canbe induced to differentiate to form a wide variety of cells, includingcells of all three embryonic lineages, mesodermal, endodermal, andectodermal. These methods have been published and are described in U.S.and PCT applications referenced above, which are herein incorporated byreference in their entirety, particularly as to methods for isolatingMAPCs, culturing them, inducing them to differentiate, and expandingboth undifferentiated and differentiated MAPCs in vitro. The ability todifferentiate MAPCs in vitro reflects the multipotency of these cells invivo, where experiments have shown that they can form virtually all (ifnot all) cells of an organism.

In particular, the ability of MAPCs to differentiate in vitro into cellsof hematopoietic lineage and immune cells has been well demonstrated.

Approaches for Transplantation to Prevent Immune Rejection

a. Universal Donor Cells:

MAPCs have cell surface profiles consistent with evasion of immunerecognition, and in their natural state may not stimulate immunesensitization and rejection. They may serve as natural universal donorcells even if their progeny mature to cells which ordinarily would beimmune-recognized and rejected.

Alternatively, MAPCs also can be manipulated to serve as universal donorcells. MAPCs can be modified to serve as universal donor cells byeliminating HLA-type I and HLA-type II antigens, and potentiallyintroducing the HLA-antigens from the prospective recipient to avoid thecells becoming easy targets for NK-mediated killing, or becomingsusceptible to unlimited viral replication and/or malignanttransformation. Elimination of HLA-antigens can be accomplished byhomologous recombination or via introduction of point-mutations in thepromoter region or by introduction of a point mutation in the initialexon of the antigen to introduce a stop-codon, such as withchimeroplasts. Transfer of the host HLA-antigen can be achieved byretroviral, lentiviral, adeno associated virus or other viraltransduction or by transfection of the target cells with the HLA-antigencDNAs. MAPCs can be used to establish a set amount or a given range orlevel of a protein in the body or blood.

b. Gene Therapy:

MAPCs can be extracted and isolated from the body, grown in culture inthe undifferentiated state or induced to differentiate in culture, andgenetically altered using a variety of techniques, especially viraltransduction. Uptake and expression of genetic material is demonstrable,and expression of foreign DNA is stable throughout development.Retroviral and other vectors for inserting foreign DNA into stem cellsare known to those of skill in the art. (Mochizuki, H., et al. (1998) J.Virol 72(11): 8873-8883; Robbins, P., et al. (1997) J. Virol. 71(12):9466-9474; Bierhuizen, M., et al. (1997) Blood 90(9): 3304-3315;Douglas, J., et al. (1999) Hum. Gene Ther. 10(6): 935-945; Zhang, G., etal. (1996) Biochem. Biophys. Res. Commun. 227(3): 707-711). Oncetransduced using a retroviral vector, enhanced green fluorescent protein(eGFP) expression persists in terminally differentiated muscle cells,endothelium, and c-Kit positive cells derived from the isolated MAPCs,demonstrating that expression of retroviral vectors introduced into MAPCpersists throughout differentiation. Terminal differentiation wasinduced from cultures initiated with 10 eGFP+ cells previouslytransduced by retroviral vector and sorted a few weeks into the initialMAPC culture period.

Genetically-Modified Stem Cells as Described in PCT/US00/21387

MAPCs can be genetically altered ex vivo, eliminating one of the mostsignificant barriers for gene therapy. For example, a subject's bonemarrow aspirate is obtained, and from the aspirate MAPCs are isolated.The MAPCs are then genetically altered to express one or more desiredgene products. The MAPCs can then be screened or selected ex vivo toidentify those cells which have been successfully altered, and thesecells can be reintroduced into the subject, either locally orsystemically. Alternately, MAPCs can be genetically altered and culturedto induce differentiation to form a specific cell lineage fortransplant. In either case, the transplanted MAPCs provide astably-transfected source of cells that can express a desired geneproduct. Especially where the patient's own bone marrow aspirate is thesource of the MAPCs, this method provides an immunologically safe methodfor producing transplant cells.

Methods for Genetically Altering MAPCs as Described in PCT/US00/21387

Cells isolated by the method described herein can be geneticallymodified by introducing DNA or RNA into the cell by a variety of methodsknown to those of skill in the art. These methods are generally groupedinto four major categories: (1) viral transfer, including the use of DNAor RNA viral vectors, such as retroviruses (including lentiviruses),Simian virus 40 (SV40), adenovirus, Sindbis virus, and bovinepapillomavirus for example; (2) chemical transfer, including calciumphosphate transfection and DEAE dextran transfection methods; (3)membrane fusion transfer, using DNA-loaded membranous vesicles such asliposomes, red blood cell ghosts, and protoplasts, for example; and (4)physical transfer techniques, such as microinjection, electroporation,or direct “naked” DNA transfer. MAPCs can be genetically altered byinsertion of pre-selected isolated DNA, by substitution of a segment ofthe cellular genome with pre-selected isolated DNA, or by deletion of orinactivation of at least a portion of the cellular genome of the cell.Deletion or inactivation of at least a portion of the cellular genomecan be accomplished by a variety of means, including but not limited togenetic recombination, by antisense technology (which can include theuse of peptide nucleic acids, or PNAs), or by ribozyme technology, forexample. Insertion of one or more pre-selected DNA sequences can beaccomplished by homologous recombination or by viral integration intothe host cell genome. The desired gene sequence can also be incorporatedinto the cell, particularly into its nucleus, using a plasmid expressionvector and a nuclear localization sequence. Methods for directingpolynucleotides to the nucleus have been described in the art. Thegenetic material can be introduced using promoters that will allow forthe gene of interest to be positively or negatively induced usingcertain chemicals/drugs, to be eliminated following administration of agiven drug/chemical, or can be tagged to allow induction by chemicals(including but not limited to the tamoxifen responsive mutated estrogenreceptor) expression in specific cell compartments (including but notlimited to the cell membrane).

Homologous Recombination as Described in PCT/US00/21387

Calcium phosphate transfection, which relies on precipitates of plasmidDNA/calcium ions, can be used to introduce plasmid DNA containing atarget gene or polynucleotide into isolated or cultured MAPCs. Briefly,plasmid DNA is mixed into a solution of calcium chloride, then added toa solution which has been phosphate-buffered. Once a precipitate hasformed, the solution is added directly to cultured cells. Treatment withDMSO or glycerol can be used to improve transfection efficiency, andlevels of stable transfectants can be improved usingbis-hydroxyethylamino ethanesulfonate (BES). Calcium phosphatetransfection systems are commercially available (e.g., ProFection® fromPromega Corp., Madison, Wis.).

DEAE-dextran transfection, which is also known to those of skill in theart, may be preferred over calcium phosphate transfection wheretransient transfection is desired, as it is often more efficient.

Since the MAPCs are isolated cells, microinjection can be particularlyeffective for transferring genetic material into the cells. Briefly,cells are placed onto the stage of a light microscope. With the aid ofthe magnification provided by the microscope, a glass micropipette isguided into the nucleus to inject DNA or RNA. This method isadvantageous because it provides delivery of the desired geneticmaterial directly to the nucleus, avoiding both cytoplasmic andlysosomal degradation of the injected polynucleotide. This technique hasbeen used effectively to accomplish germline modification in transgenicanimals.

MAPCs can also be genetically modified using electroporation. The targetDNA or RNA is added to a suspension of cultured cells. The DNA/RNA-cellsuspension is placed between two electrodes and subjected to anelectrical pulse, causing a transient permeability in the cell's outermembrane that is manifested by the appearance of pores across themembrane. The target polynucleotide enters the cell through the openpores in the membrane, and when the electric field is discontinued, thepores close in approximately one to 30 minutes.

Liposomal delivery of DNA or RNA to genetically modify the cells can beperformed using cationic liposomes, which form a stable complex with thepolynucleotide. For stabilization of the liposome complex, dioleoylphosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPC)can be added. A recommended reagent for liposomal transfer isLipofectin® (Life Technologies, Inc.), which is commercially available.Lipofectin®, for example, is a mixture of the cationic lipidN-[1-(2,3-dioleyloyx)propyl]-N—N—N-trimethyl ammonia chloride and DOPE.Delivery of linear DNA, plasmid DNA, or RNA can be accomplished eitherin vitro or in vivo using liposomal delivery, which may be a preferredmethod due to the fact that liposomes can carry larger pieces of DNA,can generally protect the polynucleotide from degradation, and can betargeted to specific cells or tissues. A number of other deliverysystems relying on liposomal technologies are also commerciallyavailable, including Effectene™ (Qiagen), DOTAP (Roche MolecularBiochemicals), FuGene 6™ (Roche Molecular Biochemicals), andTransfectam® (Promega). Cationic lipid-mediated gene transfer efficiencycan be enhanced by incorporating purified viral or cellular envelopecomponents, such as the purified G glycoprotein of the vesicularstomatitis virus envelope (VSV-G), in the method of Abe, A., et al. (J.Virol. (1998) 72: 6159-6163).

Gene transfer techniques which have been shown effective for delivery ofDNA into primary and established mammalian cell lines usinglipopolyamine-coated DNA can be used to introduce target DNA into MAPCs.This technique is generally described by Loeffler, J. and Behr, J.(Methods in Enzymology (1993) 217: 599-618).

Naked plasmid DNA can be injected directly into a tissue mass formed ofdifferentiated cells from the isolated MAPCs. This technique has beenshown to be effective in transferring plasmid DNA to skeletal muscletissue, where expression in mouse skeletal muscle has been observed formore than 19 months following a single intramuscular injection. Morerapidly dividing cells take up naked plasmid DNA more efficiently.Therefore, it is advantageous to stimulate cell division prior totreatment with plasmid DNA.

Microprojectile gene transfer can also be used to transfer genes intoMAPCs either in vitro or in vivo. The basic procedure formicroprojectile gene transfer was described by J. Wolff (GeneTherapeutics (1994) at page 195). Briefly, plasmid DNA encoding a targetgene is coated onto microbeads, usually 1-3 micron sized gold ortungsten particles. The coated particles are placed onto a carrier sheetinserted above a discharge chamber. Once discharged, the carrier sheetis accelerated toward a retaining screen. The retaining screen forms abarrier which stops further movement of the carrier sheet while allowingthe polynucleotide-coated particles to be propelled, usually by a heliumstream, toward a target surface, such as a tissue mass formed ofdifferentiated MAPCs. Microparticle injection techniques have beendescribed previously, and methods are known to those of skill in the art(see Johnston, S. A., et al. (1993) Genet. Eng. (NY) 15: 225-236;Williams, R. S., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 2726-2730;Yang, N. S., et al. (1990) Proc. Natl. Acad. Sci. USA 87: 9568-9572).

Signal peptides can be attached to plasmid DNA, as described bySebestyen, et al. (Nature Biotech. (1998) 16: 80-85), to direct the DNAto the nucleus for more efficient expression.

Viral vectors are used to genetically alter MAPCs and their progeny.Viral vectors are used, as are the physical methods previouslydescribed, to deliver one or more target genes, polynucleotides,antisense molecules, or ribozyme sequences, for example, into the cells.Viral vectors and methods for using them to deliver DNA to cells arewell known to those of skill in the art. Examples of viral vectors whichcan be used to genetically alter the cells of the present inventioninclude, but are not limited to, adenoviral vectors, adeno-associatedviral vectors, retroviral vectors (including lentiviral vectors),alphaviral vectors (e.g., Sindbis vectors), and herpes virus vectors.

Retroviral vectors are effective for transducing rapidly-dividing cells,although a number of retroviral vectors have been developed toeffectively transfer DNA into non-dividing cells as well (Mochizuki, H.,et al. (1998) J. Virol. 72: 8873-8883). Packaging cell lines forretroviral vectors are known to those of skill in the art. Packagingcell lines provide the viral proteins needed for capsid production andvirion maturation of the viral vector. Generally, these include the gag,pol, and env retroviral genes. An appropriate packaging cell line ischosen from among the known cell lines to produce a retroviral vectorwhich is ecotropic, xenotropic, or amphotropic, providing a degree ofspecificity for retroviral vector systems.

A retroviral DNA vector is generally used with the packaging cell lineto produce the desired target sequence/vector combination within thecells. Briefly, a retroviral DNA vector is a plasmid DNA which containstwo retroviral LTRs positioned about a multicloning site and SV40promoter so that a first LTR is located 5′ to the SV40 promoter, whichis operationally linked to the target gene sequence cloned into themulticloning site, followed by a 3′ second LTR. Once formed, theretroviral DNA vector can be transferred into the packaging cell lineusing calcium phosphate-mediated transfection, as previously described.Following approximately 48 hours of virus production, the viral vector,now containing the target gene sequence, is harvested.

Targeting of retroviral vectors to specific cell types was demonstratedby Martin, F., et al., (J. Virol. (1999) 73: 6923-6929), who used asingle-chain variable fragment antibody directed against the surfaceglycoprotein high-molecular-weight melanoma-associated antigen fused tothe amphotropic murine leukemia virus envelope to target the vector todeliver the target gene to melanoma cells. Where targeted delivery isdesired, as, for example, when differentiated cells are the desiredobjects for genetic alteration, retroviral vectors fused to antibodyfragments directed to the specific markers expressed by each celllineage differentiated from the MAPCs can be used to target delivery tothose cells.

Lentiviral vectors are also used to genetically alter MAPCs. Many suchvectors have been described in the literature and are known to those ofskill in the art (Salmons, B. and Gunzburg, W. H. (1993) Hum. GeneTherapy 4: 129-141. These vectors have been effective for geneticallyaltering human hematopoietic stem cells (Sutton, R., et al. (1998) J.Virol. 72: 5781-5788). Packaging cell lines have been described forlentivirus vectors (see Kafri, T., et al. (1999) J. Virol. 73: 576-584;Dull, T., et al. (1998) J. Virol. 72: 8463-8471).

Recombinant herpes viruses, such as herpes simplex virus type I (HSV-1)have been used successfully to target DNA delivery to cells expressingthe erythropoietin receptor (Laquerre, S., et al. (1998) J. Virol. 72:9683-9697). These vectors can also be used to genetically alter theMAPCs, which the inventors have demonstrated to be stably transduced bya viral vector.

Adenoviral vectors have high transduction efficiency, can incorporateDNA inserts up to 8 Kb, and can infect both replicating anddifferentiated cells. A number of adenoviral vectors have been describedin the literature and are known to those of skill in the art (see, forexample, Davidson, B. L., et al. (1993) Nature Genetics 3: 219-223;Wagner, E., et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6099-6103).Methods for inserting target DNA into an adenovirus vector are known tothose of skill in the art of gene therapy, as are methods for usingrecombinant adenoviral vectors to introduce target DNA into specificcell types (Wold, W. (1998) Humana Methods in Molecular Medicine,Blackwell Science, Ltd.). Binding affinity for certain cell types hasbeen demonstrated by modification of the viral vector fiber sequence.Adenovirus vector systems have been described which permit regulatedprotein expression in gene transfer (Molin, M., et al. (1998) J. Virol.72: 8358-8361). A system has also been described for propagatingadenoviral vectors with genetically modified receptor specificities toprovide transductional targeting to specific cell types (Douglas, J., etal. (1999) Nature Biotech. 17: 470-475). Recently described ovineadenovirus vectors even address the potential for interference withsuccessful gene transfer by preexisting humoral immunity (Hofmann, C.,et al. (1999) J. Virol. 73: 6930-6936).

Adenovirus vectors are also available which provide targeted genetransfer and stable gene expression using molecular conjugate vectors,constructed by condensing plasmid DNA containing the target gene withpolylysine, with the polylysine linked to a replication-incompetentadenovirus. (Schwarzenberger, P., et al. (1997) J. Virol. 71:8563-8571.)

Alphavirus vectors, particularly the Sindbis virus vectors, are alsoavailable for transducing the cells of the present invention. Thesevectors are commercially available (Invitrogen, Carlsbad, Calif.) andhave been described in, for example, U.S. Pat. No. 5,843,723, as well asby Xiong, C., et al. (1989) Science 243: 1188-1191; Bredenbeek, P. J.,et al. (1993) J. Virol. 67: 6439-6446; and Frolov, I., et al. (1996)Proc. Natl. Acad. Sci. USA 93: 11371-11377.

The inventors have shown that MAPCs possess good transduction potentialusing the eGFP-MND lentiviral vector described by Robbins, et al. (J.Virol. (1997) 71(12): 9466-9474) and eGFP-MGF vector. Using this method,30-50% of MAPC can be transduced after a short exposure of 4.6 hours toan enhanced green fluorescent protein (eGFP) vector containingsupernatants made in PA3-17 packaging cells (an amphotropic packagingcell line derived from NIH 3T3 fibroblasts and described by Miller, A.D., and C. Buttimore (Mol. Cell. Biol. (1986) δ: 2895-2902), combinedwith protamine (8 mg/ml). Expression of eGFP persists throughout theculture of undifferentiated MAPCs. In addition, transfection usinglipofectamine has been successfully used to introduce transgenes inMAPCs.

Successful transfection or transduction of target cells can bedemonstrated using genetic markers, in a technique that is known tothose of skill in the art. The green fluorescent protein of Aequoreavictoria, for example, has been shown to be an effective marker foridentifying and tracking genetically modified hematopoietic cells(Persons, D., et al. (1998) Nature Medicine 4: 1201-1205). Alternativeselectable markers include the β-Gal gene, the truncated nerve growthfactor receptor, and drug selectable markers (including but not limitedto NEO, MTX, and hygromycin).

Any of these techniques can also be applied to introducing atranscriptional regulatory sequence into MAPCs to activate a desiredendogenous gene. This can be done by both homologous (e.g., U.S. Pat.No. 5,641,670) or non-homologous (e.g., U.S. Pat. No. 6,602,686)recombination. These are incorporated by reference for teaching ofmethods of endogenous gene activation.

The present invention is additionally described by way of the followingillustrative, non-limiting examples.

EXAMPLES Example 1: Human MAPCs (from Bone Marrow Mononuclear Cells)

Bone marrow mononuclear cells were obtained from bone marrow aspiratesfrom the posterior iliac crest of >80 healthy human volunteers. Ten to100 cubic centimeters of bone marrow was obtained from each subject, asshown in Table 2, which indicates the approximate number of mononuclearcells isolated from each subject. Mononuclear cells (“MNCs”) wereobtained from bone marrow by centrifugation over a Ficoll-Paque densitygradient (Sigma Chemical Co., St Louis, Mo.). Bone marrow MNCs wereincubated with CD45 and Glycophorin A microbeads (Miltenyi Biotec,Sunnyvale, Calif.) for 15 minutes and CD45⁺GlyA⁺ cells removed byplacing the sample in front of a SuperMACS magnet. The eluted cells are99.5% CD45⁻GlyA⁻.

As shown in Table 2, depletion of CD45⁺GlyA⁺ cells resulted in recoveryof CD45⁻ GlyA⁻ cells which constituted approximately 0.05 to 0.10% ofthe total bone marrow mononuclear cells.

TABLE 2 Number of MAPCs in Human Bone Marrow Estimated by LDA Number ofNumber of Number of MAPCs Volume of Mononuclear BM CD45⁻ GlyA⁻(estimated by Bone Marrow Cells Post Ficoll Cells Post- limitingdilution (cc) (in millions) SuperMACS assay, LDA) 50 100 100,000 50 2580 60,000 35 25 50 14,000 10 50 100 50,000 30 10 150 75,000 30 30 100100,000 25 25 80 75,000 35 100 190 78,000 25 100 150 60,000 15 100 160160,000 85 100 317 400,000 50 100 200 150,000 70 50 160 160,000 85 50115 150,000 70 25 60 60,000 30 100 307 315,000 100 100 216 140,000 80 50130 150,000 40 100 362 190,000 60 50 190 150,000 40 100 200 185,000 100100 387 300,000 170 50 100 130,000 20 150 588 735,000 300

Cells were selected that do not express the common leukocyte antigenCD45, or the erythroid precursor marker glycophorin-A (GlyA). CD45⁻GlyA⁻cells constitute 1/10³ marrow mononuclear cells. CD45⁻GlyA⁻ cells wereplated in wells coated with fibronectin in 2% FCS, EGF, PDGF-BB,dexamethasone, insulin, linoleic acid, and ascorbic acid. After 7-21days, small clusters of adherent cells developed. Using limitingdilution assays, the frequency of cells giving rise to these adherentclusters is ⅕×10³ CD45⁻GlyA⁻ cells.

When colonies appeared (about 10³ cells), cells were recovered bytrypsinization and re-plated every 3-5 days at a 1:4 dilution under thesame culture conditions. Cell densities were maintained between 2-8×10³cells/cm². Cell doubling time was 48-60 h. Immunophenotypic analysis byFACS of cells obtained after 10-12 cell doubling showed that cells didnot express CD31, CD34, CD36, CD38, CD45, CD50, CD62E and CD62-P, Muc18,cKit, Tie/Tek, and CD44. Cells expressed no HLA-DR or HLA-class-I andexpressed low levels of β2-microglobulin. Cells stained highly positivewith antibodies against CD10, CD13, CD49b, CD49e, CDw90, and Flk1. TheMAPC phenotype remained unchanged for >30 cell doublings (n=15). MAPCcultures with cells capable of proliferating beyond 30 cell doublingsand differentiating to all mesodermal cell-types have been establishedfrom >85% of donors, age 2-50 years. In ten donors, we have expandedMAPCs for >50 cell doublings. When cells were cultured in serum-freemedium, also supplemented with 10 ng/ml IGF, cell doubling was slower(>60 h), but >40 cell doublings could be obtained. As was seen for cellscultured with 2% FCS without IGF, cells cultured in serum-free mediumwere HLA-class-I and CD44 negative, and could differentiate into allmesodermal phenotypes.

When cells were plated on collagen-type-I or laminin instead offibronectin, they expressed CD44 and HLA-DR, and could not be expandedbeyond 30 cell doublings. When EGF or PDGF were omitted, cells did notproliferate and died. Increased concentrations of these cytokinesallowed initial growth of MAPCs but caused loss of proliferation beyond20-30 cell doublings. Addition of higher concentrations of dexamethasonealso caused loss of proliferation beyond 30 cell doubling. When cellswere cultured with >2% FCS in the culture medium, they expressed CD44,HLA-DR, and HLA-class-I. Likewise, culture at high density (>8×10³cells/cm²) was associated with the acquisition of CD44, HLA-DR andHLA-class-I, and Muc-18. Culture at high density or with higherconcentrations of FCS was also associated with loss of expansioncapacity, and cells did not proliferate beyond 25-30 cell doublings.

MAPCs were replated at 1 cell/well once cultures had been established.From three donors, >2000 cells were plated singly in FN coated 96 wellplates with the same culture medium. Cell growth was not detected. Whencells were deposited at 10 cells/well, cell growth in approximately 4%of wells was detected. Progeny of 5% of these wells could be expanded to>10⁷ cells.

Telomere length of MAPCs from five donors (age 2-50 years) cultured for15 cell doublings was between 11-16 kb. In three donors, this was 3 kblonger than telomere length of blood lymphocytes obtained from the samedonors. Telomere length of cells from one donor evaluated after 15 celldoublings, 30 cells doublings, and 45 cell doublings remained unchanged.Cytogenetic analysis of MAPCs recovered after 30 cell doublings showed anormal karyotype.

Example 2: Mouse MAPCs

All tissues were obtained according to guidelines from the University ofMinnesota IACUC. BM mononuclear cells (BMMNC) were obtained by FicollHypaque separation. BM was obtained from 5-6 week old ROSA26 mice orC57/BL6 mice. Alternatively, muscle and brain tissue was obtained from3-day old 129 mice. Muscles from the proximal parts of fore and hindlimbs were excised and thoroughly minced. The tissue was treated with0.2% collagenase (Sigma Chemical Co., St Louis, Mo.) for 1 hour at 37°C., followed by 0.1% trypsin (Invitrogen, Grand Island, N.Y.) for 45minutes. Cells were then triturated vigorously and passed through a 70μm filter. Cell suspensions were collected and centrifuged for 10minutes at 1600 rpm. Brain tissue was dissected and minced thoroughly.Cells were dissociated by incubation with 0.1% trypsin and 0.1% DNAse(Sigma) for 30 minutes at 37° C. Cells were then triturated vigorouslyand passed through a 70 μm filter. Cell suspensions were collected andcentrifuged for 10 minutes at 1600 rpm.

BMMNC or muscle or brain suspensions were plated at 1×10⁵/cm² inexpansion medium [2% FCS in low glucose Dulbecco's minimal essentialmedium (LG-DMEM), 10 ng/ml each of platelet derived growth factor(PDGF), epidermal growth factor (EGF) and leukemia inhibitory factor(LIF)] and maintained at 5×10³/cm². After 3-4 weeks, cells recovered bytrypsin/EDTA were depleted of CD45⁺GlyA⁺ cells with micromagnetic beads.Resulting CD45⁻GlyA⁻ cells were replated at 10 cells/well in 96 wellplates coated with FN and were expanded at cell densities between 0.5and 1.5×10³/cm². The expansion potential of MAPCs was similar regardlessof the tissue from which they were derived.

Murine MAPCs obtained after 46 to >80 PDs were tested by Quantitative(Q)-RT-PCR for expression levels of oct-4 and rex-1, two transcriptionfactors important in maintaining an undifferentiated status of ES cells.RNA was extracted from mouse MAPCs, neuroectodermal differentiatedprogeny (day 1-7 after addition of bFGF) and mouse ES cells. RNA wasreverse transcribed and the resulting cDNA underwent 40 rounds ofamplification (ABI PRISM 7700, Perkin Elmer/Applied Biosystems) with thefollowing reaction conditions: 40 cycles of a two step PCR (95° C. for15 seconds, 60° C. for 60 seconds) after initial denaturation (95° C.for 10 minutes) with 2 μl of DNA solution, 1× TaqMan SYBR GreenUniversal Mix PCR reaction buffer. Primers are listed in Table 3.

TABLE 3 Primers Used NEO 5′-TGGATTGCACGCAGGTTCT-3′5′-TTCGCTTGGTGGTCGAATG-3′ Oct4 5′-GAAGCGTTTCTCCCTGGATT-3′5′-GTGTAGGATTGGGTGCGTT-3′ Rex1 5′-GAAGCGTTCTCCCTGGAATTTC-3′5′-GTGTAGGATTGGGTGCGTTT-3′ Otx1 5′-GCTGTTCGCAAAGACTCGCTAC-3′5′-ATGGCTCTGGCACTGATACGGATG-3′ Otx2 5′-CCATGACCTATACTCAGGCTTCAGG-3′5′-GAAGCTCCATATCCCTGGGTGGAAAG-3′ Nestin 5′ 5′-GGAGTGTCGCTTAGAGGTGC-3′5′-TCCAGAAAGCCAAGAGAAGC-3′

mRNA levels were normalized using GAPDH as housekeeping gene, andcompared with levels in mouse ES cells. Oct-4 and rex-1 mRNA werepresent at similar levels in BM, muscle, and brain derived MAPCs. Rex-1mRNA levels were similar in mMAPCs and mES cells, while oct-4 mRNAlevels were about 1,000 fold lower in MAPCs than in ES cells.

The Gene Expression Profiles of Mouse MAPCs from Bone Marrow, Muscle,and Brain are Highly Similar

To further evaluate whether MAPCs derived from different tissues weresimilar, the expressed gene profiles of BM, muscle, and brain derivedMAPCs were examined using U74A Affymetrix gene arrays. Briefly, mRNA wasextracted from 2-3×10⁶ BM, muscle, or brain derived-MAPCs cultured for45 population doublings. Preparation of cDNAs, hybridization to the U74Aarrays containing 6,000 murine genes and 6,000 EST clusters, and dataacquisition were done per manufacturer's recommendations (all fromAffymetrix, Santa Clara, Calif.). Data analysis was done using GeneChip®software (Affymetrix). Increased or decreased expression by a factor of2.2 fold (Iyer, V. R. et al. (1999) Science 283: 83-7; Scherf, U. et al.(2000) Nat. Biotech 24: 236-44; Alizadeh, A. A. et al. (2000) Nature403: 503-11) was considered significant. r2 value was determined usinglinear regression analysis.

Comparison between the expressed gene profiles of MAPCs from the threetissues showed that <1% of genes were expressed at >2.2-fold differentlevels in MAPCs from BM than muscle. Likewise, the expression of <1% ofgenes in BM derived MAPCs was >2.2-fold different than in brain derivedMAPCs. As the correlation coefficient between the different MAPCpopulations was >0.975, it was concluded that MAPCs derived from thedifferent tissues are highly homologous, in line with the phenotypic anddifferentiation characteristics described herein.

Using the mouse-specific culture conditions, mMAPC cultures have beenmaintained for more than 100 cell doublings. mMAPC cultures have beeninitiated with marrow from C57Bl/6 mice, ROSA26 mice, and C57BL/6 micetransgenic for HMG-LacZ.

Example 3: Rat MAPCs

BM and MNCs from Sprague Dawley, Lewis, or Wistar rats were obtained andplated under conditions similar to those described for mMAPCs. After21-28 days, cells were depleted of CD45⁺ cells, and the resulting CD45⁻cells were subcultured at 10 cells/well.

Similar to mMAPCs, rMAPCs have been culture expanded for >100 PDs.Expansion conditions for rat MAPC culture required the addition of EGF,PDGF-BB, and LIF, and culture on FN, but not collagen type I, laminin,or Matrigel™.

Rat MAPCs that had undergone 42 PDs, 72 PDs, 80 PDs, and 100 PDs, wereharvested and telomere lengths evaluated. Telomeres did not shorten inculture, as determined by Southern blot analysis after 42 PDs, 72 PDs,80 PDs, and 100 PDs. Monthly cytogenetic analysis of rat MAPCs revealednormal karyotype.

Example 4: Clonality of Mouse and Rat MAPCs

To demonstrate that differentiated cells were single cell-derived andMAPCs are indeed “clonal” multipotent cells, cultures were made in whichMAPCs had been transduced with a retroviral vector. UndifferentiatedMAPCs and their progeny were found to have the retrovirus inserted inthe same site in the genome.

Studies were done using two independently derived ROSA26 MAPCs, twoC57BL/6 MAPCs, and one rMAPC population expanded for 40 to >90 PDs, aswell as with the eGFP transduced “clonal” mouse and “clonal” rMAPCs. Nodifferences were seen between eGFP transduced and untransduced cells. Ofnote, eGFP expression persisted in differentiated MAPCs.

Specifically, murine and rat BMMNCs cultured on FN with EGF, PDGF-BB,and LIF for three weeks were transduced on two sequential days with aneGFP oncoretroviral vector. Afterwards, CD45⁺ and GlyA⁺ cells weredepleted and cells sub-cultured at 10 cells/well. eGFP-transduced ratBMMNCs were expanded for 85 PDs. Alternatively, mouse MAPCs expanded for80 PDS were used. Subcultures of undifferentiated MAPCs were generatedby plating 100 MAPCs from cultures maintained for 75 PDs andre-expanding them to >5×10⁶ cells. Expanded MAPCs were induced todifferentiate in vitro to endothelium, neuroectoderm, and endoderm.Lineage differentiation was shown by staining with antibodies specificfor these cell types.

Example 5: Human MAPCs are not Immunogenic

Mesenchymal stem cells have demonstrated low in vitro immunogenicity andthe ability to engraft across allogeneic recipients (Di Nicola, M. etal. (2002) Blood 99: 3838-3843; Jorgensen, C. et al. (2002) Gene Therapy10: 928-931; Le Blanc, K. et al. (2003) Scandinavian Journal ofImmunology 57: 11-20; McIntosh, K. et al. (2000) Graft 6: 324-328; Tse,W. et al. (2003) Transplantation 75: 389-397).

FIG. 3 shows that human MAPCs exhibit low in vitro immunogenicity andare immunosuppressive when added to otherwise potent T-cell MLRs (Tse,W. et al. (2003)). Results were consistent across all donor andresponder pairs tested.

Responder and stimulator cells were prepared for these experiments andthe MLRs were performed according to the procedures described by Tse, W.et al. (2003).

Example 6: MAPCs Modulate T-Cell Responses

The ability of MAPCs to modulate, and in this case suppress, immuneresponsiveness is illustrated by T-cell proliferation assays, forexample, which can be carried out as follows.

Preparation of Responder T-Cells

Responder cells were prepared from lymph nodes of Lewis rats. Lymphnodes were surgically removed from the rats and immediately placed into3 to 5 ml of media (complete RPMI or complete DMEM) in 60×15 mm Petriplates. The lymph nodes were dispersed through a nylon filter (using thehand plunger of a syringe). The dispersions were loaded into 50 ml tubesand centrifuged at 1,250 rpm for 8 minutes. The resulting supernatants(media) were discarded. The pellets (containing the cells) wereresuspended in fresh media (complete RPMI or complete DMEM). The cellswere washed three times, and then resuspended in fresh media.Resuspended cell densities were determined by counting the number ofcells in a known volume thereof. The cells were maintained on ice. Priorto use, the cells were resuspended in media (complete RPMI or completeDMEM) at a density of 1.25×10⁶ cells/ml.

Preparation of MAPCs

MAPCs were prepared from Lewis or from Sprague-Dawley rats and thenfrozen as described above. They were thawed and then irradiated at 3000rad. The irradiated cells were then resuspended in media (complete RPMIor complete DMEM) to densities of 0.4×10⁶ cells/ml, 0.8×10⁶ cells/ml,and 1.6×10⁶ cells/ml.

Preparation of Concanavalin A

Concanavalin A (“ConA”) was used to activate the T-cells. The ConA wasdissolved in PBS (complete RPMI or complete DMEM) to finalconcentrations of 0 (PBS only) 10, 30, and 100 μg/ml.

Assay Procedure

Each data point is based on at least three determinations.

20 μl of each of the ConA solutions was added to wells of microtiterplates (96 well, flat bottom), followed by 80 μl/well of the respondercells and 100 μl/well of the MAPCs. The plates were incubated at 37° C.in humidified incubators under 5% CO₂ for 4-5 days. The plates werepulsed with 1 μCi/well ³H-thymidine during the last 14-18 hours ofculture. Thereafter, the cells were automatically harvested on glassfiber filters using a Tomtec harvesting machine. The thymidine uptakewas quantified in a micro-plate scintillation counter. Results wereexpressed as mean counts per minute (CPM)+/−SD.

Final concentrations of ConA in the growth media in the wells were 0, 1,3.16, and 10 μg/ml. MAPCs were present in the wells in the amounts of 0,0.4, 0.8, and 1.6×10⁵ cells/well.

The results, described below, are illustrated in FIG. 4.

Results

Increasing amounts of ConA resulted in a dose-dependent stimulation ofT-cell proliferation (FIG. 4, LN only). Lewis MAPCs inhibitedproliferation of the ConA stimulated T-cells. The inhibition depended onthe dose of MAPCs. The maximum inhibition, 50%, occurred at the highestdose of MAPCs used in these experiments and with low and intermediatedoses of ConA. These results are displayed graphically in FIG. 4 andshow that MAPCs suppress the proliferation of activated T-cells.

Example 7: MAPCs Suppress Proliferation of Stimulated T-Cells

The ability of MAPCs to suppress proliferation of syngeneic andnon-matched (allogeneic) T-cell proliferative responses is demonstratedby the results of mixed lymphocyte reactions. The example below showsthe suppressive effects of MAPCs on T-cells from Lewis rats stimulatedby splenocytes from irradiated DA rats. MAPCs from syngeneic Lewis ratsand non-matched (allogeneic) Sprague-Dawley rats both inhibited T-cellresponses in a dose-dependent manner. The experiments were carried outas follows.

Preparation of Responder T-Cells

Responder cells were prepared from the lymph nodes of Lewis rats asdescribed above.

Irradiated Spleen Stimulator Cells

Spleens were surgically removed from DA rats. Splenocytes then wereisolated from the spleens essentially as described above for theisolation of responder cells from the lymph nodes of Lewis rats. Theisolated spleen cells were irradiated at 1800 rad. The cells then wereresuspended to 4×10⁶/ml and kept on ice until they were used.

Preparation of MAPCs

Syngeneic MAPCs were prepared from Lewis rats as described above.Non-matched (allogeneic) MAPCs were prepared from Sprague-Dawley rats inthe same way. Both Lewis and Sprague-Dawley MAPCs were irradiated at3000 rad, then resuspended in complete RPMI media at densities of0.03×10⁶/ml, 0.06×10⁶/ml, 0.125×10⁶/ml, 0.25×10⁶/ml, 0.5×10⁶/ml,1×10⁶/ml, and 2×10⁶/ml.

Assay Procedure

96 well microtiter plate wells were loaded with: 100 μl of MAPCs (at thedilutions indicated above) or 100 μl of media; 50 μl of a stimulatorcell stock or control; 50 μl each of a responder cell stock or control;and media (complete RPMI or complete DMEM) as required to equalize totalvolumes to a final volume of 200 μl/well. 96 well flat bottom microtiterplates were used for all assays.

Each data point is based on at least three determinations.

The plates were incubated at 37° C. in humidified incubators under 5%CO₂ for 4-5 days. The plates were pulsed with 1 μCi/well ³H-thymidineduring the last 14-18 hours of culture. Thereafter, the cells wereautomatically harvested on glass fiber filters using a Tomtec harvestingmachine. The thymidine uptake was quantified in a micro-platescintillation counter. Results were expressed as mean counts per minute(CPM)+/−SD.

Results

Exposure of T-cells derived from the lymph nodes of Lewis rats(Responders) to stimulator cells consisting of irradiated splenocytesfrom DA rats (Stimulators) resulted in very robust proliferativeresponses of responder cells, as shown in FIGS. 5A and 5B, for “noMAPC”.

Addition of increasing doses of syngeneic Lewis MAPCs (FIG. 5A) andnon-matched (allogeneic) third-party Sprague-Dawley MAPCs (FIG. 5B)resulted in a significant and dose-dependent inhibition of T-cellactivation. Maximal levels of inhibition were ˜80%. Even at the lowestdoses of MAPCs, there was 40-50% inhibition.

There was no ³H-thymidine incorporation in the controls, showing thatincorporation was due solely to proliferation of activated responderT-cells, as shown in FIGS. 5A and 5B.

In summary, the results show that syngeneic and third-party (allogeneic)MAPCs suppress T-cell proliferation even in the presence of potentactivator splenocytes from non-matched rats. In these experiments,inhibition peaked when there were similar numbers (200,000 cells) eachof stimulators, responders, and MAPCs in the reaction. Under theseconditions, there was ˜80% inhibition. There was very substantialinhibition at much lower ratios of MAPCs to the other cells in thereaction. For instance, at 1.5% MAPCs, there was 50% inhibition (3,000MAPCs versus 200,000 of each of the types of cells). The resultsdemonstrate not only that MAPCs have a strong immunosuppressive effect,but also that a relatively small number of MAPCs is sufficient toinhibit a relatively large number of competent T-cells even in thepresence of very potent T-cell activators.

Example 8: MAPCs are Safe

The main immediate risk of intravenous injection of large numbers ofcells is the accumulation of cell clumps in the lungs which results inrespiratory distress and can cause cardiac arrest. To show the safety ofMAPCs in this regard, we measured the effects of intravenous injectionof MAPCs on respiratory rates in Buffalo rats.

MAPCs were prepared from Lewis rats as described above (“Lewis MAPCs”).Splenocytes also were prepared from Lewis rats as described above foruse as controls (“Lewis Splenocytes”).

One female Lewis rat served as the splenocyte donor for each group(experimental condition). Two female Buffalo rats were used asrecipients for each group (experimental condition).

The cells were administered to the rats as indicated below. The data aredisplayed graphically in FIG. 6. The concordance of data points to theindividual rats as numbered below is enumerated in the vertical legendat the right of FIG. 6. All rats were Buffalo rats.

1.1, 1.2 10 × 10⁶ Lewis MAPC per rat 2.1, 2.2 5 × 10⁶ Lewis MAPC per rat3.1, 3.2 2.5 × 10⁶ Lewis MAPC per rat 4.1, 4.2 1.2 × 10⁶ Lewis MAPC perrat 5.1, 5.2 10 × 10⁶ Lewis Splenocytes per rat 6.1, 6.2 5 × 10⁶ LewisSplenocytes per rat 7.1, 7.2 2.5 × 10⁶ Lewis Splenocytes per rat 8.1,8.2 1.2 × 10⁶ Lewis Splenocytes per rat

As indicated above, rats were injected with 1.2, 2.5, 5, or 10 millionMAPCs or 1.2, 2.5, 5, or 10 million splenocytes. This corresponded to 5,10, 25, or 50 million cells/kg. Respiratory rates were measured before(0 min) and at 1, 5, and 10 min after intravenous injection of the MAPCsor splenocytes. Respiratory rates were measured for 20 seconds and thecounts were multiplied by 3 to derive the per minute respiratory rates.Normal rat respiratory rates are between 60 and 140/min.

Results

All animals survived after intravenous cell injections. No significantdifferences or trends were observed in the respiratory rates under thedifferent conditions. The results are shown in FIG. 6. Initialrespiratory rates (0 min) were slightly reduced because the animals wereanesthetized before the cells were injected. At each time-point,measurements were clustered without any apparent trends.

In summary, intravenous injection of 5-50 million MAPCs/kg did not causechanges in respiratory rates or mortality in any of the rats under anyof the conditions. The results show that intravenous injection of MAPCsis safe even at high doses.

Example 9: MAPC Expression of Immune Markers

The immunomodulatory nature of MAPCs was further characterized bydetermining immune regulatory markers in MAPCs, such as those describedby Barry et al. (2005). The markers were determined usingmarker-specific antibodies and FACS analysis.

Rat bone marrow MAPCs were isolated, cultured, and harvested asdescribed above. For FACS analysis, the cells were suspended at 1-2×10⁸cells/ml in PBS. 200 μl of the cell suspension was added to each of aseries of 12×75 polypropylene tubes. A marker-specific antibody or acontrol was added to each of the tubes, and they were then incubated for15-20 minutes at room temperature. At the end of the incubation period,2 ml of PBS was added to each tube and they were then centrifuged at400×g for 5 minutes. Supernatants were discarded and the cells wereresuspended in each tube in 100 μl of PBS. A fluorescent labeledsecondary antibody was added to each tube in an appropriate volume, andthe tubes were again incubated for 15-20 minutes at room temperature,this time in the dark. Thereafter, 2 ml of PBS was added to each tubeand they were again centrifuged at 400×g for 5 minutes. Supernatantswere discarded, and the cells in each tube were resuspended in 200 μl ofPBS and were then kept on ice until analyzed by FACS.

Results are enumerated and presented graphically in Table 4. As shown inthe table, rat MAPCs are: (a) positive for MHC class I, CD11c, CD29, andCD90, and (b) negative for MHC class II, CD11b, CD31, CD40, CD45, CD54,CD80, CD86, CD 95, and CD106. Negative results were validated for eachantibody by positive staining of control cells, including rat peripheralblood and endothelial cells. These patterns of marker expression, as toboth the markers that are detected in MAPCs and those that were notdetected, are fully consistent with the low immunostimulatorycross-section of MAPCs.

TABLE 4 Detection of Immune Cell-Related Markers in MAPCs Name/Functionof Commercial Anti- FACS Marker Marker Rat-Marker Antibody Detection MHCclass I yes Positive MHC class II yes Negative CD11b Mac-1 yes NegativeCD11c Integrin αX yes Positive CD29 Integrin β1 yes Positive CD31PECAM-1 yes Negative CD40 TNFRSF yes Negative CD45 Leucocyte common yesNegative antigen CD54 ICAM-1 yes Negative CD80 B7-1 yes Negative CD86B7-2 yes Negative CD90 Thy-1 yes Positive CD95 Apo-1 yes Negative CD106VCAM-1 yes Negative

Example 10: MAPCs Suppress Previously Stimulated T-Cells in MLRs

Cells

Responder cells were prepared from the lymph nodes of Lewis rats asdescribed above. Splenocytes, prepared from Buffalo or DA rats, asdescribed above, were used as stimulators. MAPCs were prepared asdescribed above.

Procedures

MAPCs were added to a first group of MLRs at the same time assplenocytes (as was done in the foregoing examples). In addition, MAPCswere added to a second group of MLRs that were set-up identically to thefirst group. However, the MAPCs in the second group were added 3 daysafter the addition of the splenocytes (or control). Thus, in the firstgroup, MAPCs were added before the T-cells began proliferating inresponse to the splenocytes. In the second group, the MAPCs were addedwhen the T-cell response to the splenocytes had been underway for 3days. All plates were incubated for a total of 4 days, then pulsed with³H-thymidine and harvested, as described in the foregoing examples. Theexperiments otherwise were carried out as described for the MLRs in theforegoing examples.

Each data point is based on at least three determinations.

Results

As can be seen from the right side of FIG. 7, MAPCs strongly suppressT-cell proliferation in MLRs when they are added three days afterstimulation by allogeneic splenocytes. Comparison of the left and rightsides of FIG. 7 shows that MAPCs strongly suppress the on-goingproliferation reaction. Quantitatively, the results show for Buffalocells that MAPCs added 3 days post-stimulation suppressed T-cellproliferation 50% compared to controls (right side of the figure), and75% when added at the same time as the stimulatory splenocytes (leftside of the figure). Similarly, for DA cells, MAPCs added 3 dayspost-stimulation suppressed T-cell proliferation 33% compared tocontrols (right side of the figure), and 70% when added at the same timeas the stimulatory splenocytes (left side of the figure).

In all cases, the degree of immunosuppression by MAPCs depended, ingeneral, on the number of MAPCs that were added to the reaction. Inother words, immunosuppression depended on the dose of MAPCs added tothe MLRs.

Publications cited above are incorporated herein by reference in theirentirety as to specific subject matter for which they have been cited.

What is claimed is:
 1. A method of treatment of diabetes in a subject,comprising: administering to a subject in need of treatment fordiabetes—by an effective route and in an effective amount to treatdiabetes, cells that: are not embryonic stem cells, embryonic germcells, or germ cells; can differentiate into at least one cell type ofeach of at least two of the endodermal, ectodermal, and mesodermalembryonic lineages; express telomerase; are allogeneic or xenogeneic tothe subject; do not provoke a deleterious immune response in thesubject; and are effective to treat diabetes in the subject,—wherein thecells are administered adjunctively to one or more other treatments,without also administering an immunosuppressive treatment adjunctivelyto treatment with the cells wherein the cells have not been geneticallyengineered to improve their immunomodulatory properties.
 2. A methodaccording to claim 1, wherein said cells can differentiate into at leastone cell type of each of the endodermal, ectodermal, and mesodermalembryonic lineages.
 3. A method according to claim 1, wherein said cellsare positive for oct-3/4.
 4. A method according to claim 1, wherein saidcells have undergone at least 10 to 40 cell doublings in culture priorto their administration to the subject.
 5. A method according to claim1, wherein said cells are allogeneic to the subject.
 6. A methodaccording to claim 1, wherein said cells are administered to the subjectadjunctively to another treatment that is administered before, at thesame time as, or after said cells are administered.
 7. A methodaccording to claim 1, wherein said cells are mammalian cells.
 8. Amethod according to claim 7, wherein said cells are human cells.
 9. Amethod according to claim 7, wherein said cells are derived from cellsisolated from placental tissue, umbilical cord tissue, umbilical cordblood, bone marrow, blood, spleen tissue, thymus tissue, spinal cordtissue, or liver tissue.
 10. A method according to claim 1, wherein thesubject is a mammal.
 11. A method according to claim 10, wherein thesubject is human.
 12. A method according to claim 11, wherein said cellsare administered to the subject in one or more doses comprising 10⁴ to10⁸ of said cells per kilogram of the subject's mass.
 13. A methodaccording to claim 1, wherein the diabetes is Type I diabetes mellitus.14. A method according to claim 13, wherein said cells can differentiateinto at least one cell type of each of the endodermal, ectodermal, andmesodermal embryonic lineages.
 15. A method according to claim 13,wherein said cells are positive for oct-3/4.
 16. A method according toclaim 13, wherein said cells have undergone at least 10 to 40 celldoublings in culture prior to their administration to the subject.
 17. Amethod according to claim 13, wherein said cells are allogeneic to thesubject.
 18. A method according to claim 13, wherein said cells aremammalian cells.
 19. A method according to claim 13, wherein said cellsare human cells.
 20. A method according to claim 13, wherein said cellsare derived from cells isolated from placental tissue, umbilical cordtissue, umbilical cord blood, bone marrow, blood, spleen tissue, thymustissue, spinal cord tissue, or liver tissue.
 21. A method according toclaim 13, wherein the subject is a mammal.
 22. A method according toclaim 13, wherein the subject is human.
 23. A method according to claim13, wherein said cells are administered to the subject in one or moredoses comprising 10⁴ to 10⁸ of said cells per kilogram of the subject'smass.
 24. A method according to claim 13, wherein the diabetes is Type Idiabetes mellitus.
 25. A method according to claim 1, wherein the cellsare derived from bone marrow.
 26. A method according to claim 25,wherein the cells are derived from human bone marrow.
 27. A methodaccording to claim 13, wherein the cells are derived from bone marrow.28. A method according to claim 27, wherein the cells are derived fromhuman bone marrow.